Near-infrared dyes as surface enhanced raman scattering reporters

ABSTRACT

Nanoparticles comprising surface-enhanced Raman scattering (SERS) reporter molecules of the formula A-Y and methods of their use are disclosed, wherein A is: 
     
       
         
         
             
             
         
       
     
     wherein X 1  is CR 4  or N; and Y is selected from the group consisting of:

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 12/134,594, filed on Jun. 6, 2008 which claims the benefit of thefiling date of U.S. Provisional Patent Application No. 60/942,329 filedon Jun. 6, 2007, the disclosures of which are hereby incorporated hereinby reference.

BACKGROUND OF THE INVENTION

The presently disclosed subject matter relates to near-infrared dyes andtheir use as surface-enhanced Raman scattering (SERS) reportermolecules.

When a molecule is irradiated with photons of a particular frequency,the photons are scattered. The majority of the incident photons areelastically scattered without a change in frequency (Rayleighscattering), whereas a small fraction of the incident photons(approximately 1 in every 10⁸) interact with a vibrational mode of theirradiated molecule and are inelastically scattered. The inelasticallyscattered photons are shifted in frequency and have either a higherfrequency (anti-Stokes) or a lower frequency (Stokes). By plotting thefrequency of the inelastically scattered photons against theirintensity, a unique Raman spectrum of the molecule is observed. The lowsensitivity of conventional Raman spectroscopy, however, has limited itsuse for characterizing biological samples in which the target analyte(s)typically are present in small quantities.

When a Raman-active molecule is adsorbed on or in close proximity to,e.g., within about 50 Å of, a metal surface, the intensity of a Ramansignal arising from the Raman-active molecule can be enhanced. Thisenhancement is referred to as the surface-enhanced Raman scattering(SERS) effect. The SERS effect was first reported in 1974 by Fleishmanet al., who observed intense Raman scattering from pyridine adsorbed ona roughened silver electrode surface. See Fleishman et al., “Ramanspectra of pyridine adsorbed at a silver electrode,” Chem. Phys. Lett.,26, 163 (1974); see also Jeanmaire, D. L., and Van Dyne, R. P., “SurfaceRaman spectroelectrochemistry. 1. Heterocyclic, aromatic, andaliphatic-amines absorbed on anodized silver electrode.” J. Electroanal.Chem., 84(1), 1-20 (1977); Albrecht, M. G., and Creighton, J. A.,“Anonymously intense Raman spectra of pyridine at a silver electrode,”J.A.C.S., 99, 5215-5217 (1977). Since then, SERS has been observed for anumber of different molecules adsorbed on the surface of metal surfaces.See, e.g., A. Campion, A. and Kambhampati, P., “Surface-enhanced Ramanscattering,” Chem. Soc. Rev., 27, 241 (1998).

The magnitude of the SERS enhancement depends on a number of parameters,including the position and orientation of various bonds present in theadsorbed molecule with respect to the electromagnetic field at the metalsurface. The mechanism by which SERS occurs is thought to result from acombination of (i) surface plasmon resonances in the metal that enhancethe local intensity of the incident light; and (ii) formation andsubsequent transitions of charge-transfer complexes between the metalsurface and the Raman-active molecule.

The SERS effect can be observed with Raman-active molecules adsorbed onor in close proximity to metal colloidal particles, metal films ondielectric substrates, and metal particle arrays, including metalnanoparticles. For example, Kneipp et al. reported the detection ofsingle molecules of a dye, cresyl violet, adsorbed on aggregatedclusters of colloidal silver nanoparticles. See Kneipp, K. et al.,“Single molecule detection using surface-enhanced Raman scattering(SERS), Phys. Rev. Lett., 78(9), 1667-1670 (1997). That same year, Nieand Emory observed the surfaced enhanced resonance Raman spectroscopy(SERRS) signal, wherein the resonance between the absorption energy ofthe Raman-active molecule and that of the nanoparticle yield anenhancement as large as about 10¹⁰ to about 10¹², of a dye moleculeadsorbed on a single silver nanoparticle, where the nanoparticles rangedfrom spherical to rod-like and had a dimension of about 100 nm. See Nie,S., and Emory, S. R., “Probing single molecules and single nanoparticlesby surface-enhanced Raman scattering,” Science, 275, 1102-1106 (1997);Emory, S. R., and Nie, S., “Near-field surface-enhanced Ramanspectroscopy on single silver nanoparticles,” Anal. Chem., 69, 2631(1997).

Even with the enhanced signal due to the SERS or SERRS effect, the useof Raman spectroscopy can be limited in diagnostic assays andapplications requiring a high sensitivity. Accordingly, there is a needin the art for SERS-active reporter molecules that give rise to anincreased Raman signal when compared to SERS-active reporter moleculesknown in the art. The presently disclosed subject matter addresses, inwhole or in part, these and other needs in the art.

BRIEF SUMMARY OF THE INVENTION

In some embodiments, the presently disclosed subject matter provides ananoparticle comprising a surface enhanced Raman scattering(SERS)-active reporter molecule of the formula:

A-Y

wherein:

A is selected from the group consisting of:

wherein X₁ is CR₄ or N;

Y is selected from the group consisting of:

wherein:

r, s, and t are each independently an integer from 1 to 8;

each X₂ and X₃ is independently selected from the group consisting of C,S, and N, under the proviso that (i) when X₂ is C or S, R₅ is Z, or whenX₃ is C or S, R₆ is Z, as Z is defined herein below; (ii) if both X₂ andX₃ are N at the same time, at least one of R₅ and R₆ is absent; and(iii) when X₂ is N, R₅ when present is Z′, or when X₃ is N, R₆ whenpresent is Z′, wherein Z′ is selected from the group consisting of:

(CH₂)_(n)—X₄; —NR₈—(CH₂)_(p)—X₅; —(CH₂)_(q)X₆C(═O)—R₉,

wherein:

n, p, q, u, and v are each independently an integer from 1 to 8;

X₄ and X₅ are each independently selected from the group consisting ofhydroxyl, amino, and thiol;

X₆ is O or NR₁₁;

wherein:

each R₁, R₂, R₃, R₄, R₅, R₆, R₈, R₁₀, R₁₁, and Z is independentlyselected from the group consisting of H, alkyl, substituted alkyl,heteroalkyl, substituted heteroalkyl, cycloalkyl, substitutedcycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl,substituted aryl, aralkyl, hydroxyl, alkoxyl, hydroxyalkyl,hydroxycycloalkyl, alkoxycycloalkyl, aminoalkyl, acyloxyl,alkylaminoalkyl, and alkoxycarbonyl;

R₇ is Z′;

R₉ is —(CH₂)_(m)—X₇ or —(CH₂)_(m)—B, wherein

m is an integer from 1 to 8;

X₇ is halogen; and

B is a binding member having a binding affinity for a ligand or analyteto be detected.

In some embodiments, the presently disclosed subject matter provides amethod for labeling a molecule, cell, bead, or solid support, the methodcomprising: (a) providing at least one of a molecule, cell, bead, orsolid support; and (b) attaching a particle to the at least one of amolecule, cell, bead, or solid support, wherein the particle comprises asurface-enhanced Raman spectroscopy (SERS)-active nanoparticle havingassociated therewith a dye of Formula A-Y.

In some embodiments, the presently disclosed subject matter provides amethod for detecting the presence or amount of one or more analytes in abiological sample, the method comprising: (a) providing a biologicalsample suspected of containing one or more analytes; (b) contacting thebiological sample with a reagent comprising one or more SERS-activenanoparticles having associated therewith at least one specific bindingmember having an affinity for the one or more analytes and at least oneSERS-active reporter molecule of Formula A-Y; (c) illuminating thebiological sample with incident radiation at a wavelength to induce theSERS-active reporter molecule to produce a SERS signal; and (d)measuring the SERS signal to detect the presence or amount of one ormore analytes in the biological sample.

In some embodiments, the presently disclosed subject matter provides amethod for detecting the presence of one or more target structures in asample cell, the method comprising: (a) contacting one or more samplecells with one or more SERS-active nanoparticles labeled with one ormore binding members under conditions suitable for binding of the one ormore binding members to one or more target structures in the samplecell, wherein the SERS-active nanoparticle has associated therewith adye of Formula A-Y capable of producing a distinguishable Raman signal;and (b) detecting one or more distinguishable SERS signals from thesample cell to indicate the presence of the one or more targetstructures in the sample cell.

In some embodiments, the presently disclosed subject matter provides akit including a reagent comprising one or more surface-enhanced Ramanspectroscopy (SERS)-active nanoparticles having associated therewith atleast one SERS-active reporter molecule of Formula A-Y.

Thus, it is an object of the presently disclosed subject matter toprovide a nanoparticle comprising a SERS-active reporter molecule ofFormula A-Y. It is another object of the presently disclosed subjectmatter to provide a method for labeling a molecule, cell, bead, or solidsupport, with a nanoparticle comprising a SERS-active reporter moleculeof Formula A-Y. It is yet another object of the presently disclosedsubject matter to provide a method for detecting the presence or amountof one or more analytes in a biological sample. It is another object ofthe presently disclosed subject matter to provide a method for detectingthe presence of one or more target structures in a sample cell. It isanother object of the presently disclosed subject matter to provide akit including a reagent comprising one or more surface-enhanced Ramanspectroscopy (SERS)-active nanoparticles having associated therewith atleast one SERS-active reporter molecule of Formula A-Y.

Certain objects of the presently disclosed subject matter having beenstated hereinabove, which are addressed in whole or in part by thepresently disclosed subject matter, other objects will become evident asthe description proceeds when taken in connection with the accompanyingExamples and Drawings as best described herein below.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus described the presently disclosed subject matter in generalterms, reference will now be made to the accompanying drawings, whichare not necessarily drawn to scale, and wherein:

FIG. 1 shows a Raman spectrum obtained from a Raman reporter moleculeknown in the art, e.g., trans-1,2-bis(4-pyridyl)ethylene (BPE) (dottedline) and a Raman spectrum of a presently disclosed near-infrared dye,e.g., coumarin picolinium (CoPic);

FIG. 2 shows a comparison of the Raman intensity observed withnon-fluorescent Raman molecules and commercial dyes adsorbed on 60-nmspherical gold nanoparticles. FIG. 2A shows the Raman intensity ofnon-fluorescent Raman molecules adsorbed on 60-nm gold nanoparticles.FIG. 2B shows the Raman intensity of commercial dyes adsorbed on 60-nmspherical gold nanoparticles;

FIG. 3 shows a comparison of the Raman intensity of commercial Ramanreporter molecules and commercial dyes and the presently disclosednear-infrared dyes adsorbed on spherical gold nanoparticles;

FIG. 4 shows chemical structures of representative embodiments of thepresently disclosed near-infrared dyes;

FIG. 5 is a schematic showing DNA probes immobilized to magneticparticle (M) and a SERS-active nanoparticle via a polyethylene glycol(PEG) linker according to one embodiment of the presently disclosedsubject matter (only one probe shown for clarity and not drawn toscale); and

FIGS. 6A-6C show representative SERS spectra from the presentlydisclosed magnetic capture assays;

FIG. 6A shows the SERS spectra of an empty sample tube (solid line) andof oligonucleotide-coated SERS-active nanoparticles associated witholigonucleotide-coated magnetic particles (dashed line) in the absenceof target DNA, wherein the oligonucleotide has been attached directly tothe SERS-active nanoparticles and the magnetic particles, respectively,through biotin-streptavidin associations; and

FIGS. 6B and 6C show representative SERS spectra ofoligonucleotide-coated SERS-active nanoparticles associated witholigonucleotide-coated magnetic particles in the absence of target DNA,wherein the oligonucleotides have been attached to the SERS-activenanoparticles and the magnetic particles, respectively, via apolyethylene glycol linker molecule. FIG. 6C presents the same data asFIG. 6B on a more narrow scale. The dashed line in FIG. 6C representsthe SERS spectrum of an empty sample tube and the solid lines representthe assay signal.

DETAILED DESCRIPTION

The presently disclosed subject matter now will be described more fullyhereinafter with reference to the accompanying Drawings, in which some,but not all embodiments of the presently disclosed subject matter areshown. Many modifications and other embodiments of the presentlydisclosed subject matter set forth herein will come to mind to oneskilled in the art to which the presently disclosed subject matterpertains having the benefit of the teachings presented in the foregoingdescriptions and the associated Drawings. Therefore, it is to beunderstood that the presently disclosed subject matter is not to belimited to the specific embodiments disclosed and that modifications andother embodiments are intended to be included within the scope of theappended claims. Although specific terms are employed herein, they areused in a generic and descriptive sense only and not for purposes oflimitation.

The terms “a,” “an,” and “the” refer to “one or more” when used in thisapplication, including the claims. Thus, for example, reference to “asample” includes a plurality of samples, unless the context clearly isto the contrary (e.g., a plurality of samples), and so forth.

Throughout this specification and the claims, the words “comprise,”“comprises,” and “comprising” are used in a non-exclusive sense, exceptwhere the context requires otherwise.

As used herein, the term “about,” when referring to a value is meant toencompass variations of, in some embodiments±50%, in someembodiments±20%, in some embodiments±10%, in some embodiments±5%, insome embodiments±1%, in some embodiments±0.5%, and in someembodiments±0.1% from the specified amount, as such variations areappropriate to perform the disclosed methods or employ the disclosedcompositions.

I. Nanoparticles Comprising Near-IR SERS-Active Reporter Molecules

The presently disclosed subject matter provides near-infrared dyes andtheir use as SERS-active reporter molecules, also referred to herein asSERS-active molecules, SERS-active dyes, or SERS-active labels. A“reporter molecule” refers to any molecule or chemical compound that iscapable of producing a Raman spectrum when it is illuminated withradiation of a proper wavelength. A “reporter molecule” also can bereferred herein as a “label,” a “dye,” a “Raman-active molecule,” or“SERS-active molecule,” each of which can be used interchangeably.

As described in more detail herein below, the intensity of the SERSsignal observed for the presently disclosed SERS-active dyes whenassociated with, i.e., adsorbed on or attached to, a nanoparticle ishigher than that observed for SERS-active reporter molecules andcommercial dyes known in the art. The enhanced SERS signals observed forthe presently disclosed SERS-active dyes allow for their use inapplications, such as diagnostic assays using Raman spectroscopy as adetection method, optical imaging of tissues and cells, and otherapplications, where high sensitivity is required.

When used in a diagnostic assay, the enhanced SERS signals observed forthe presently disclosed SERS-active dyes enable detection of biomarkers,including, but not limited to, proteins, nucleic acids, and metabolites,at lower concentrations than those measurable using SERS-active reportermolecules known in the art. This higher sensitivity is beneficial inapplications where the Raman signal has to pass through, i.e., istransmitted through, a complex medium, such as whole blood or serum.Further, diagnostic assays with a higher sensitivity for analytes ofinterest can be required for early detection of a condition or a diseasestate in a subject.

A. Nanoparticles Comprising Near-IR SERS-Active Reporter Molecules ofFormula A-Y

In some embodiments, the presently disclosed subject matter provides ananoparticle comprising a SERS-active reporter molecule of the Formula:

A-Y

wherein:

A is selected from the group consisting of:

wherein X₁ is CR₄ or N;

Y is selected from the group consisting of:

wherein:

r, s, and t are each independently an integer from 1 to 8;

each X₂ and X₃ is independently selected from the group consisting of C,S, and N, under the proviso that (i) when X₂ is C or S, R₅ is Z, or whenX₃ is C or S, R₆ is Z, as Z is defined herein below; (ii) if both X₂ andX₃ are N at the same time, at least one of R₅ and R₆ is absent; and(iii) when X₂ is N, R₅ when present is Z′, or when X₃ is N, R₆ whenpresent is Z′, wherein Z′ is selected from the group consisting of:

—(CH₂)_(n)—X₄; —NR₈—(CH₂)_(p)—X₅; —(CH₂)_(q)X₆C(═O)—R₉,

wherein:

n, p, q, u, and v are each independently an integer from 1 to 8;

X₄ and X₅ are each independently selected from the group consisting ofhydroxyl, amino, and thiol;

X₆ is O or NR₁₁;

wherein:

each R₁, R₂, R₃, R₄, R₅, R₆, R₈, R₁₀, R₁₁, and Z is independentlyselected from the group consisting of H, alkyl, substituted alkyl,heteroalkyl, substituted heteroalkyl, cycloalkyl, substitutedcycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl,substituted aryl, aralkyl, hydroxyl, alkoxyl, hydroxyalkyl,hydroxycycloalkyl, alkoxycycloalkyl, aminoalkyl, acyloxyl,alkylaminoalkyl, and alkoxycarbonyl;

R₇ is Z′;

R₉ is —(CH₂)_(m)—X₇ or —(CH₂)_(m)—B, wherein

m is an integer from 1 to 8;

X₇ is halogen; and

B is a binding member having a binding affinity for a ligand or analyteto be detected.

In some embodiments, the variable “A” of formula A-Y comprises acoumarin nucleus, an aza-coumarin nucleus, a benzoxadiazole nucleus, oranalogs or derivatives thereof. Representative structures of a coumarinnucleus, an aza-coumarin nucleus, and a benzoxadiazole nucleus areprovided immediately herein below:

As used herein, an “analog” refers to a chemical compound in which oneor more individual atoms or functional groups of a parent compound havebeen replaced, either with a different atom or with a differentfunctional group. For example, thiophene is an analog of furan, in whichthe oxygen atom of the five-membered furanyl ring is replaced by asulfur atom.

As used herein, a “derivative” refers to a chemical compound that isderived from or obtained from a parent compound and contains essentialelements of the parent compound, but typically has one or more differentfunctional groups. Such functional groups can be added to a parentcompound, for example, to improve the molecule's solubility, absorption,biological half life, Raman activity, and the like, or to decrease thetoxicity of the molecule, eliminate or attenuate any undesirable sideeffect of the molecule, and the like. A derivative of a parent compoundis meant to include any chemical modification, addition, deletion, orsubstitution to the parent compound. In some embodiments, a derivativeof a parent compound can include any reaction product of the derivative,for example, the reaction product of the derivative with an amino acidresidue. Accordingly, in some embodiments, the presently disclosedSERS-active nanoparticle can include a dye of Formula A-Y, wherein thedye nucleus includes a reactive group that can be conjugated, e.g.,covalently attached, to an amino acid, for example, an amino acidresidue of a protein. A non-limiting example of a derivative is an esteror amide of a parent compound having a carboxylic acid functional group.

In some embodiments, the SERS-active reporter dye molecule of FormulaA-Y is selected from the group consisting of:

wherein X′ includes hydroxyl, amino, and thiol; and R₁ and R₂ are asdefined hereinabove.

In some embodiments, the presently disclosed SERS-active nanoparticlescan be conjugated with a specific binding member “B” of a binding pair.The specific binding member can be conjugated with the SERS-activereporter molecule, e.g., a compound of formula A-Y through, for example,a thiol group as represented by the variable X′, or bound to orotherwise associated with the nanoparticle itself. As used herein, aspecific binding member is a member of a specific binding pair. A“specific binding pair” refers to two different molecules, where one ofthe molecules through chemical or physical means specifically binds thesecond molecule. In this sense, an analyte is a reciprocal member of aspecific binding pair. Further, specific binding pairs can includemembers that are analogs of the original specific binding partners, forexample, an analyte-analog having a similar structure to the analyte. By“similar” it is intended that, for example, an analyte-analog has anamino acid sequence that has at least about 60% or 65% sequenceidentity, about 70% or 75% sequence identity, about 80% or 85% sequenceidentity, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater amino acid sequence identity compared to an analyte amino acidsequence using alignment programs and standard parameters well known inthe art. An analog of an analyte also can have the same function as ananalyte.

In some embodiments, the binding member is a binding protein. As usedherein, the term “binding protein” refers to a protein, that whenconjugated with a SERS-active nanoparticle, interacts with a specificanalyte or ligand in a manner capable of producing a detectable Ramansignal differentiable from when a target analyte or ligand is present orabsent, or when a target analyte or ligand is present in varyingconcentrations over time. The term “producing a detectable signal”refers to the ability to recognize a change in a property of a reportergroup, e.g., a presently disclosed dye, in a manner that enables thedetection of binding member-analyte, e.g., binding protein-ligand,binding. Further, the producing of a detectable signal can be reversibleor non-reversible. The signal-producing event includes continuous,programmed, and episodic means, including one-time or reusableapplications. The reversible signal-producing event can be instantaneousor can be time-dependent, so long as a correlation with the presence orconcentration of the analyte is established.

Such embodiments can be used as a biosensor. As used herein, the terms“biosensor” and “biosensor compound” generally refer to a compound thatundergoes a detectable change in specific response to a ligand or targetanalyte. More particularly, the presently disclosed biosensors combinethe molecular recognition properties of biological macromolecules, suchas a binding protein, with SERS-active nanoparticles that produce a SERSsignal upon ligand binding.

The presently disclosed dye of Formula A-Y can be associated with, e.g.,adsorbed, or attached, e.g., covalently bound, to a nanoparticle.Generally, the term “associated” refers to a state of two molecules or amolecule and a particle, such as a nanoparticle, being held in closeproximity to one another. As provided in more detail in the Examples,the presently disclosed dyes can be mixed in an appropriate ratio with ananoparticle solution for a few hours, e.g., four to six hours, wherebythe dye is adsorbed on the nanoparticle surface. Without wishing to bebound to any one particular theory, it is believed that, in someembodiments, the presently disclosed dyes are associated with ananoparticle through the quaternary nitrogen on the pyridinium cation,which is represented by the variable “Y.”

The presently disclosed dyes can be functionalized so that the dye willbind covalently to a nanoparticle. Such embodiments can improve thestability of the dye in complex media, such as blood and serum. As usedherein, the term “direct attachment” can, in some embodiments, refer tothe covalent attachment of a SERS-active reporter molecule to thenanoparticle surface. Indirect attachment can be accomplished using anintervening compound, molecule, or the like. In some embodiments, thepresently disclosed SERS-active reporter molecule can be modified toinclude a linker, such as a thiol-containing moiety, for example, thevariable X′ as described hereinabove, which can be directly attached tothe nanoparticles, e.g., gold nanoparticles, by methods known in the artor, as disclosed in more detail herein below, a polyethylene glycol(PEG) linker. Other methods of associating the SERS-active reportermolecule, including non-covalent attachment methods known to those ofordinary skill in the art, also can be used.

A Raman enhancing nanoparticle having associated therewith, e.g.,adsorbed on or attached to, a SERS-active molecule(s) is referred toherein as a SERS-active nanoparticle. More particularly, a SERS-activenanoparticle, as referred to herein, includes a nanoparticle have asurface that induces, causes, or otherwise supports surface-enhancedRaman light scattering (SERS) or surface-enhanced resonance Raman lightscattering (SERRS). A number of surfaces are capable of producing a SERSsignal, including roughened surfaces, textured surfaces, and othersurfaces, including smooth surfaces.

“Raman scattering” generally refers to the inelastic scattering of aphoton incident on a molecule. Photons that are inelastically scatteredhave an optical frequency (ν_(i)), which is different than the frequencyof the incident light (ν₀). The difference in energy (ΔE) between theincident light and the inelastically scattered light can be representedas (ΔE)=h|ν₀−ν_(i)|, wherein h is Planck's constant, and corresponds toenergies that are absorbed by the molecule. The incident radiation canbe of any frequency ν₀, but typically is monochromatic radiation in thevisible spectral region. The absolute difference |ν₀−ν_(i)| is aninfrared, e.g., vibrational, frequency. The process that produces lightof frequency other than ν₀ is referred to as “Raman scattering.” Thefrequency ν₁ of the “Raman scattered” radiation can be greater than orless than ν₀, but the amount of light with frequency ν₁<ν₀ (Stokesradiation) is greater than that with frequency ν₁>ν₀ (anti-Stokesradiation).

As used herein, the term “radiation” refers to energy in the form ofelectromagnetic radiation that can induce surface-enhanced Ramanscattering in a sample under test, e.g., a sample comprising aSERS-active nanoparticle having one or more of the presently disclosedSERS-active reporter molecules associated therewith. More particularly,the term “radiation” refers to energy in the form of electromagneticradiation that causes the surface of a nanoparticle to induce, emit,support, or otherwise cause light scattering, e.g., Raman scattering, ina reporter molecule proximate to the nanoparticle surface.

“Surface-enhanced Raman scattering” or “SERS” refers to the phenomenonthat occurs when the Raman scattering signal, or intensity, is enhancedwhen a Raman-active molecule is adsorbed on or in close proximity to,e.g., within about 50 Å of, a metal surface. Under such circumstances,the intensity of the Raman signal arising from the Raman-active moleculecan be enhanced. “Surface-enhanced resonance Raman scattering” or“SERRS” refers to an increased SERS signal that occurs when the reportermolecule in close proximity to the SERS-active nanoparticle surface isin resonance with the excitation wavelength.

As used herein, the terms “nanoparticle,” “nanostructure,”“nanocrystal,” “nanotag,” and “nanocomponent,” are used interchangeablyand refer to a particle having at least one dimension in the range ofabout 1 nm to about 1000 nm, including any integer value between 1 nmand 1000 nm (including about 1, 2, 5, 10, 20, 50, 60, 70, 80, 90, 100,200, 500, and 1000 nm). In some embodiments, the nanoparticle is ametallic nanoparticle. In some embodiments, the nanoparticle is aspherical particle, or substantially spherical particle having a corediameter between about 2 nm and about 200 nm (including about 2, 5, 10,20, 50, 60, 70, 80, 90, 100, and 200 nm). In some embodiments, thenanoparticle has a core diameter between about 2 nm and about 100 nm(including about 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 nm)and in some embodiments, between about 20 nm and 100 nm (including about20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, and 100 nm). One of ordinary skill inthe art, upon review of the presently disclosed subject matter, wouldrecognize that a nanoparticle suitable for use with the presentlydisclosed assays can include a core, e.g., a metal core, which inducesthe Raman effect, and can further include one or more layers ofSERS-active materials, encapsulants, and/or outer shell structures thatalso can contribute to the size, e.g., total diameter of thenanoparticle structure.

SERS-active nanoparticles suitable for use with the presently discloseddyes typically comprise at least one metal, i.e., at least one elementselected from the Periodic Table of the Elements that is commonly knownas a metal. Suitable metals include Group 11 metals, such as Cu, Ag, andAu, or any other metals known by those skilled in the art to supportSERS, such as alkali metals. In some embodiments, the nanoparticlesubstantially comprises a single metal element. For example, thepreparation of gold nanoparticles is described by Frens, G., Nat. Phys.Sci., 241, 20 (1972). In other embodiments, the nanoparticle comprises acombination of at least two elements, such as an alloy, for example, abinary alloy. In some embodiments, the nanoparticle is magnetic.

In other embodiments, the metal includes an additional component, suchas in an Au₂S/Au core-shell particle. Au₂S/Au core-shell particles havebeen reported to have widely tunable near-IR optical resonance. SeeAveritt, R. D., et al., “Ultrafast optical properties of goldnanoshells,” JOSA B, 16(10), 1824-1832 (1999). Further, Ag core/Au shellparticles, such as those described by Cao, Y. W., et al., “DNA-modifiedcore-shell Ag/Au nanoparticles,” J. Am. Chem. Soc., 123(32), 7961-7962(2001), or Au core/Ag shell particles, or any core-shell combinationinvolving SERS-active metals, can be used. Other combinations suitablefor use in core-shell particles also are suitable for use with thepresently disclosed subject matter, including Au- or Ag-functionalizedsilica/alumina colloids, Au- or Ag-functionalized TiO₂ colloids, Aunanoparticle capped-Au nanoparticles (see, e.g., Mucic, et al.,“DNA-directed synthesis of binary nanoparticle network materials,” J.Am. Chem. Soc., 120(48), 12674 (1998)); Au nanoparticle-capped TiO₂colloids; and particles having a Si core with a metal shell (i.e.,“nanoshells”), such as silver-capped SiO₂ colloids or gold-capped SiO₂colloids. See, e.g., Jackson, et al., Proc. Natl. Acad. Sci. U.S.A.101(52):17930-5 (2004); see also U.S. Pat. Nos. 6,344,272 and 6,685,986to Oldenburg et al., each of which is incorporated herein by referencein its entirety. The use of such nanoshells in biosensing applicationshas been described. See U.S. Pat. No. 6,699,724 to West et al., which isincorporated herein by reference in its entirety.

Another class of nanoparticles suitable for use with the presentlydisclosed SERS-active reporter molecules includes nanoparticles havingan internal surface. Such nanoparticles include hollow particles andhollow nanocrystals or porous or semi-porous nanoparticles. See, e.g.,U.S. Pat. No. 6,913,825 to Ostafin et al., which is incorporated hereinby reference in its entirety. Accordingly, the presently disclosedsubject matter also provides a nanoparticle comprising a core-shellparticle active for SERS or a hollow nanoparticle active for SERS. Insome embodiments, such nanoparticles can exhibit an improved SERSsignal.

While it is recognized that particle shape and aspect ratio can affectthe physical, optical, and electronic characteristics of nanoparticles,the specific shape, aspect ratio, or presence/absence of internalsurface area does not bear on the qualification of a particle as ananoparticle. Accordingly, nanoparticles suitable for use with thepresently disclosed dyes can have a variety of shapes, sizes, andcompositions. Further, the nanoparticle can be solid, or in someembodiments, as described immediately hereinabove, hollow. Non-limitingexamples of suitable nanoparticles include colloidal metal hollow orfilled nanobars, magnetic, paramagnetic, conductive or insulatingnanoparticles, synthetic particles, hydrogels (colloids or bars), andthe like. It will be appreciated by one of ordinary skill in the artthat nanoparticles can exist in a variety of shapes, including but notlimited to spheroids, rods, disks, pyramids, cubes, cylinders,nanohelixes, nanosprings, nanorings, rod-shaped nanoparticles,arrow-shaped nanoparticles, teardrop-shaped nanoparticles,tetrapod-shaped nanoparticles, prism-shaped nanoparticles, and aplurality of other geometric and non-geometric shapes.

Further, nanoparticles suitable for use with the presently discloseddyes can be isotropic or anisotropic. As referred to herein, anisotropicnanoparticles have a length and a width. In some embodiments, the lengthof an anisotropic nanoparticle is the dimension parallel to the aperturein which the nanoparticle was produced. In some embodiments, theanisotropic nanoparticle has a diameter (width) of about 350 nm or less.In other embodiments, the anisotropic nanoparticle has a diameter(width) of about 250 nm or less and in some embodiments, a diameter(width) of about 100 nm or less. In some embodiments, the width of theanisotropic nanoparticle is between about 15 nm to about 300 nm.Further, in some embodiments, the anisotropic nanoparticle has a length,wherein the length is between about 10 nm and 350 nm.

Much of the SERS literature (both experimental and theoretical) suggeststhat anisotropic particles (rods, triangles, prisms) can provide anincreased enhancement of the Raman signal as compared to spheres. Forexample, the so-called “antenna effect” predicts that Raman enhancementis expected to be larger at areas of higher curvature. Many reports ofanisotropic particles have been recently described, including silver(Ag) prisms and “branched” gold (Au) particles.

Anisotropic Au and Ag nanorods can be produced by electrodeposition intopreformed alumina templates, in a manner similar to the production ofNanobarcodes® particles (Oxonica Inc., Mountain View, Calif.). See,e.g., Nicewarner-Pena, S. R., et al., “Submicrometer metallic barcodes,”Science, 294, 137-141 (2001); Walton, I. D., et al., “Particles formultiplexed analysis in solution: detection and identification ofstriped metallic particles using optical microscopy,” Anal. Chem. 74,2240-2247 (2002). These particles can be prepared by the deposition ofalternating layers of materials, typically Au and Ag, into preformedalumina templates, and can have a diameter of about 250 nm and a lengthof about 6 microns.

The presently disclosed SERS-active nanoparticles also are suitable foruse in composite nanostructures, e.g., satellite structures andcore-shell structures, as disclosed in PCT International PatentApplication No. PCT/US2008/057700 to Weidemaier et al., filed Mar. 20,2008, which is incorporated herein by reference in its entirety.

B. Functionalized SERS-Active Nanoparticles

Further, SERS-active nanoparticles comprising the presently disclosedSERS-active dyes can be functionalized with a molecule, such as aspecific binding member of a binding pair, which can bind to a targetanalyte. Upon binding the target analyte, the SERS spectrum of theSERS-active reporter molecule changes in such a way that the presence oramount of the target analyte can be determined. The use of afunctionalized SERS-active nanoparticle has several advantages overnon-functionalized nanoparticles. First, the functional group provides adegree of specificity to the nanoparticle by providing a specificinteraction with a target analyte. Second, the target analyte does nothave to be Raman active itself; its presence can be determined byobserving changes in the SERS spectrum of the Raman-active dye attachedto the nanoparticle. Such measurements are referred to herein as“indirect detection,” in which the presence or absence of a targetanalyte or ligand in a biological sample is determined by detecting aSERS signal that does not directly emanate from the target analyte orligand of interest.

The presently disclosed SERS-active nanoparticles can be functionalizedto bind to a target analyte in at least two different ways. In someembodiments, the SERS-active reporter molecule, i.e., the SERS-activedye, can be conjugated with a specific binding member of a binding pair,whereas in other embodiments, a specific binding member of a bindingpair can be attached directly to the nanoparticle. In embodiments inwhich the nanoparticle core is at least partially surrounded by anencapsulating shell, the binding member can be attached to an outersurface of the encapsulating shell.

1. Functionalized SERS-Active Nanoparticles Associated with SERS-ActiveReporter Molecules Conjugated with Specific Binding Members of a BindingPair

In some embodiments, the SERS-active near-IR dye associated with aSERS-active nanoparticle can be functionalized to bind to a targetanalyte. Such functionalized SERS-active nanoparticles can be used incombination with binding assays to detect physiologically importantmolecules, including metabolites, such as glucose, lactate, and fattyacids, in biological samples. In some embodiments, the presentlydisclosed SERS-active dyes include a reactive group that can be used tocouple or conjugate the dye with another molecule, including a member ofa specific binding pair, such as a binding protein or a receptor, whichhas an affinity for a specific ligand or analyte.

In some embodiments, the dye nucleus can include a thiol-reactive groupthat can be conjugated to the thiol moiety of a cysteine amino acidresidue in a natural or an engineered or mutated protein. As usedherein, the term “thiol-reactive group” refers to a substituent groupthat can react with a thiol moiety to form a carbon-sulfur bond.Examples of suitable thiol-reactive groups that can be introduced intothe presently disclosed dyes include a halo-acetyl group and ahalo-acetamide group. In some embodiments, the halo-acetyl groupincludes an iodoacetyl group, whereas the halo-acetamide group caninclude an iodoacetamide or bromoacetamide group. One of ordinary skillin the art upon review of the presently disclosed subject matter wouldrecognize that other thiol-reactive groups known in the art, such asmaleimide groups, are suitable for use with the presently disclosedsubject matter.

As used herein, the term “conjugate” refers to a molecule comprising twoor more subunits bound together, optionally through a linking group, toform a single molecular structure. The binding can be made either by adirect chemical bond between the subunits or through a linking group.Such binding in a conjugate typically is irreversible. As used herein,the term “affinity” refers to the strength of the attraction between onebinding member to another member of a binding pair at a particularbinding site. The term “specificity” and derivations thereof, refer tothe likelihood that a binding member will bind to another member of abinding pair. Such binding between one binding member, e.g., a bindingprotein, to another binding member of a binding pair, e.g., a ligand oranalyte, can be reversible.

The term “specific binding member” refers to a molecule for which thereexists at least one separate, complementary binding molecule. A specificbinding member is a molecule that binds, attaches, or otherwiseassociates with a specific molecule. The binding, attachment, orassociation can be chemical or physical. A specific molecule to which aspecific binding member binds can be any of a variety of molecules,including, but not limited to, antigens, haptens, proteins,carbohydrates, nucleotide sequences, nucleic acids, amino acids,peptides, enzymes, and the like. Further, a specific binding member of aparticular type will bind a particular type of molecule. In suchinstances, the specific binding members are referred to as a “specificbinding pair.” Accordingly, an antibody will specifically bind anantigen. Other specific binding pairs include avidin and biotin,carbohydrates and lectins, complementary nucleotide sequences,complementary peptide sequences, enzymes and enzyme cofactors, and thelike.

Representative specific binding members of a binding pair are disclosedin more detail herein below.

2. SERS-Active Nanoparticles Having a Specific Binding Member of aBinding Pair Attached Directly Thereto

In some embodiments, a binding member of a specific binding pair, forexample, an antibody, such as a monoclonal antibody, can be attacheddirectly to the surface of the nanoparticle or to the outer surface of ashell encapsulating the nanoparticle. In an exemplary embodiment, aspecific binding member of a binding pair, e.g., a monoclonal antibody,can be treated with linker, e.g., polyethylene glycol (PEG), andattached directly to the nanoparticle through the PEG linker. Use of alinker, such as a PEG linker, allows the native properties and structureof the specific binding member to be retained and increases thespecificity of the functionalized nanoparticle by sterically hinderingnon-specific binding of other species to the nanoparticle. SERS-activenanoparticles having a specific binding member attached through a PEGlinker are described in more detail herein (see Section I.D., hereinbelow).

Depending on the binding member, one of ordinary skill in the art wouldrecognize upon review of the presently disclosed subject matter thatlinkers other than PEG can be used. For example, alkanethiols can beused as linkers for antibodies and peptides. Short chain alkanethiols,including, but not limited to, N-succinimidyl-S-acetylthioacetate (SATA)and N-succinimidyl-S-acetylthiopropionate (SATP) can be used as linkersafter sulfhydryl deprotection. Other properties also can determine thechoice of linker, such as the length of the linker chain. For example,PEG can be desirable in that it also acts to protect the surface of thereagent and is flexible, which can enhance the ability of the reagent tobind to the analyte of interest.

Nanoparticles having one or more SERS-active reporter molecules andspecific binding members attached thereto can be disposed in anappropriate medium, e.g., a buffered solution, to provide a SERS-activereagent.

3. Representative Binding Members

In some embodiments, the binding member conjugated with the presentlydisclosed SERS-active nanoparticle, either through the SERS-activereporter molecule or directly attached to an outer surface of thenanoparticle itself, comprises a polypeptide or protein. Representativebinding proteins suitable for use with the presently disclosedSERS-active nanoparticles, include, but are not limited to periplasmicbinding proteins (PBPs). Examples of PBPs include, but are not limitedto, glucose-galactose binding protein (GGBP), maltose binding protein(MBP), ribose binding protein (RBP), arabinose binding protein (ABP),dipeptide binding protein (DPBP), glutamate binding protein (GluBP),iron binding protein (FeBP), histidine binding protein (HBP), phosphatebinding protein (PhosBP), glutamine binding protein (QBP), oligopeptidebinding protein (OppA), or derivatives thereof, as well as otherproteins that belong to the families of proteins known as periplasmicbinding protein like I (PBP-like I) and periplasmic binding protein likeII (PBP-like II). For other binding proteins suitable for use with thepresently disclosed SERS-active nanoparticles, see U.S. PatentApplication Publication Nos. 2006/0078908 and 2006/0280652 to Pitner etal. and U.S. patent application Ser. No. 11/738,442, filed Apr. 20,2007, each of which is incorporated herein by reference in its entirety.

Other examples of proteins that can comprise the binding membersinclude, but are not limited to intestinal fatty acid binding proteins(FAPBs). The FABPs are a family of proteins that are expressed at leastin the liver, intestine, kidney, lungs, heart, skeletal muscle, adiposetissue, abnormal skin, adipose, endothelial cells, mammary gland, brain,stomach, tongue, placenta, testis, and retina. The family of FABPs is,generally speaking, a family of small intracellular proteins (about 14kDa) that bind fatty acids and other hydrophobic ligands throughnon-covalent interactions. See Smith, E. R. and Storch, J., J. Biol.Chem., 274 (50):35325-35330 (1999), which is incorporated herein byreference in its entirety. Members of the FABP family of proteinsinclude, but are not limited to, proteins encoded by the genes FABP1,FABP2, FABP3, FABP4, FABP5, FABP6, FABP7, FABP(9) and MP2. Proteinsbelonging to the FABP include I-FABP, L-FABP, H-FABP, A-FABP, KLBP,mal-1, E-FABP, PA-FABP, C-FABP, S-FABP, LE-LBP, DA11, LP2, MelanogenicInhibitor, and the like.

Other binding members include specific binding members having anaffinity for a target analyte, including antibodies for target analytes,such as prostate specific antigen (PSA), creatine kinase MB (CKMB)isoenzyme, cardiac troponin I (cTnI) protein, thyroid-stimulatinghormone (TSH), influenza A (Flu A) antigen, influenza B (Flu B) antigen,and respiratory syncytial virus (RSV) antigen. Antibodies for suchtarget analytes are known in the art.

As used herein, a “derivative” of a protein or polypeptide is a proteinor polypeptide that shares substantial sequence identity with thewild-type protein. Derivative proteins or polypeptides of the presentlydisclosed subject matter can be made or prepared by techniques wellknown to those of skill in the art. Examples of such techniques include,but are not limited to, mutagenesis and direct synthesis. Derivativeproteins also can be modified, either by natural processes, such aspost-translational processing, or by chemical modification techniqueswhich are well known in the art. Such modifications are well describedin basic texts and in more detailed monographs, as well as in voluminousresearch literature. Modifications can occur anywhere in the polypeptidechain, including the peptide backbone, the amino acid side-chains andthe amino or carboxyl termini. It will be appreciated that the same typeof modification can be present in the same or varying degrees at severalsites in a given polypeptide or protein. Also, a given polypeptide orprotein can contain more than one modification. Examples of derivativeproteins include, but are not limited to, mutant and fusion proteins.

A “mutant protein” is used herein as it is known in the art. In general,a mutant protein can be created by addition, deletion or substitution ofthe wild-type primary structure of the protein or polypeptide. Mutationsinclude, for example, the addition or substitution of cysteine groups,non-naturally occurring amino acids, and replacement of substantiallynon-reactive amino acids with reactive amino acids. A mutant protein canbe mutated to bind more than one analyte in a specific manner. Indeed,the mutant proteins can possess specificity towards its wild-typeanalyte and another target ligand. Likewise, a mutant protein can beable to only bind an analyte or analytes that the wild-type bindingprotein does not bind. Methods of generating mutant proteins arewell-known in the art. For example, Looger, L. L., et al., Nature 423(6936): 185-190 (2003), which is incorporated herein by reference,disclose methods for redesigning binding sites within periplasmicbinding proteins that provide new analyte-binding properties for theproteins. These mutant binding proteins retain the ability to undergoconformational change, which can produce a directly generated signalupon analyte-binding. By introducing between 5 and 17 amino acidchanges, Looger, et al. constructed several mutant proteins, each withnew selectivities for TNT (trinitrotoluene), L-lactate, or serotonin.

The mutation can serve one or more of several purposes. For example, anaturally occurring protein can be mutated to change the long-termstability, including thermal stability, of the protein, to conjugate theprotein to a particular encapsulation matrix or polymer, to providebinding sites for detectable reporter groups, to adjust its bindingconstant with respect to a particular analyte, or combinations thereof.

The analyte and mutated protein can act as binding partners. The term“associates” or “binds” as used herein refers to binding partners havinga relative binding constant (Kd) sufficiently strong to allow detectionof binding to the protein by a detection means. The Kd can be calculatedas the concentration of free analyte at which half the protein is bound,or vice versa. When the analyte of interest is glucose, the Kd valuesfor the binding partners are between about 0.0001 mM and about 50 mM.

The presently disclosed SERS-active reporter molecule can be attached toa mutated protein, for example a GGBP, by any conventional means knownin the art. For example, the reporter molecule can be conjugated to theprotein via amines or carboxyl residues on the protein. Exemplaryembodiments include covalent coupling via thiol groups on cysteineresidues of the mutated or native protein. For example, for mutatedGGBP, cysteines can be located at position 10, at position 11, position14, at position 15, position 19, at position 26, at position 43, atposition 74, at position 92, at position 93, position 107, position 110,position 112, at position 113, at position 137, at position 149, atposition 152, at position 154, at position 182, at position 183, atposition 186, at position 211, at position 213, at position 216, atposition 238, at position 240, at position 242, at position 255, atposition 257, at position 287, at position 292, at position 294, and atposition 296.

C. Encapsulated SERS-Active Nanoparticles

SERS-active metal nanoparticles have a tendency to aggregate in aqueoussolution and once aggregated are difficult to re-disperse. Further, thechemical composition of some Raman-active molecules is incompatible withchemistries used to attach other molecules, such as proteins, to metalnanoparticles. These characteristics can limit the choice ofRaman-active molecule, attachment chemistries, and other molecules to beattached to the metal nanoparticle.

Accordingly, in some embodiments, the presently disclosed SERS-activedye of Formula A-Y when affixed, e.g., either adsorbed or covalentlyattached to a nanoparticle, can be coated or encapsulated, for example,in a shell, of a different material, including a polymer, glass, orceramic material. Such embodiments are referred to herein as “compositeSERS-active nanoparticles.” Methods for preparing composite SERS-activenanoparticles are described in U.S. Pat. No. 6,514,767 to Natan, whichis incorporated herein by reference in its entirety.

The presently disclosed composite SERS-active nanoparticles can includea metal nanoparticle, a submonolayer, monolayer, or multilayer of one ormore presently disclosed dyes in close proximity to the surface of themetal nanoparticle. The term “in close proximity” is intended to meanwithin about nm or less of an outer surface of the nanoparticle. Ananoparticle having a submonolayer, monolayer, or multilayer of one ormore presently disclosed dyes attached to an outer surface of thenanoparticle core also can include an encapsulating shell. In suchembodiments, the presently disclosed dye is positioned at an interfacebetween the outer surface of the metal nanoparticle and an interiorsurface of the encapsulating shell.

The nanoparticle core comprising the composite nanoparticle can be ametal sphere, e.g., a gold, silver, or copper sphere, having a diameterof about 20 nm to about 200 nm. In some embodiments, the nanoparticlecore comprises an oblate or prolate metal spheroid. The diameter of thenanoparticle core can be selected based, in part, on the wavelength ofincident light. For example, in SERS using red incident light, i.e.,incident light having a wavelength of about 600 nm, the optimal SERSresponse is obtained with gold nanoparticle cores having a diameter ofabout 60 nm.

In some embodiments, the encapsulating shell comprises a dielectricmaterial, such as a polymer, glass, metal, metal oxides, such as TiO₂and SnO₂, metal sulfides or a ceramic material. In some embodiments, theencapsulant is glass, e.g., SiO_(x). To encapsulate the presentlydisclosed SERS-active nanoparticles in glass, the metal nanoparticlecores can be treated with a glass primer, i.e., a material that can leadto a growth of a uniform coating of glass, or can improve adhesion ofthe glass coat to the particle, or both. Glass can then be grown overthe metal nanoparticle by standard techniques known in the art.

The encapsulation process can be carried out after, or during, attachingor adsorbing one or more presently disclosed dyes to the corenanoparticle. In this way, the dye is sequestered from the surroundingsolvent as a coating on the surface of the metal nanoparticle core. Sucha configuration provides the metal nanoparticle core with a stable SERSactivity. The dye can form a sub-monolayer, a complete monolayer, or amultilayer assembly on the surface of the metal nanoparticle core. Thedye layer can comprise a single dye or can be a mixture of differentdyes.

Thus, in some embodiments, the SERS-active reporter molecule of formulaA-Y forms a layer on the outer surface of the nanoparticle core, whereinthe layer at least partially covers the outer surface of thenanoparticle core and is defined by an inner surface and an outersurface. The encapsulant is disposed on at least one of the outersurface of the nanoparticle core and the outer surface of the layer ofthe SERS-active reporter molecule of formula A-Y to at least partiallysurround the nanoparticle core, which is at least partially covered witha layer of the SERS-active reporter molecule.

Preferably, the encapsulant does not measurably alter the SERS activityof the composite SERS-active nanoparticle. The benefits of the presentlydisclosed subject matter are still achieved, however, even if theencapsulant has some measurable effect, provided it does not interferewith the SERS activity, or does not add significant complexity to theobserved SERS spectrum.

Further, in some embodiments, the encapsulant can be modified, e.g.,derivatized by standard techniques known in the art, to attachmolecules, including biomolecules, to its outer surface. Thischaracteristic allows the presently disclosed composite SERS-activenanoparticles to be conjugated to molecules, including biomolecules,such as proteins and nucleic acids, or to solid supports withoutinterfering with the Raman activity of the dye. Glass and othermaterials suitable for use as an encapsulating shell contain functionalgroups amenable to molecular attachment. For example, immersion of glassin a suitable base allows for the covalent attachment of alkyltrichlorosilanes or alkyl trialkoxysilanes, with additionalfunctionality available on the end of the alkyl group of the alkyltrichlorosilane or alkyl trialkoxysilane group. Thus, glass surfaces canbe modified with many forms of biomolecules and biomolecularsuperstructures, including cells, as well as oxides, metals, polymers,and the like. Likewise, surfaces of glass can be modified withwell-organized monomolecular layers. Accordingly, glass coatings supportmany types of chemical functionalization (also referred to herein as“derivatization”). Other forms of encapsulants also can befunctionalized, as well. Accordingly, the presently disclosednanoparticles can be affixed to any species known in the art having achemically-reactive functionality.

The thickness of the encapsulant can be varied depending on the physicalproperties required of the SERS-active nanoparticle. Depending on theparticular combination of nanoparticle core, encapsulant, and dye, thickcoatings of encapsulant, e.g., coatings on the order of one micron ormore, could potentially attenuate the Raman signal. Further, a thincoating might lead to interference in the Raman spectrum of the analyteby the molecules on the encapsulant surface. At the same time, physicalproperties, such as the sedimentation coefficient can be affected by thethickness of the encapsulant. In general, the thicker the encapsulant,the more effective the sequestration of the SERS-active dyes on themetal nanoparticle core from the surrounding solvent.

In embodiments wherein the encapsulant is glass, the thickness of theglass typically can range from about 1 nm to about 50 nm. In exemplary,non-limiting embodiments, the encapsulated SERS-active nanoparticlescomprise gold nanoparticles having a diameter ranging from about 50 nmto about 100 nm encapsulated in a sphere of glass having a thicknessranging from about, in some embodiments, from about 10 nm to about 50nm; in some embodiments, from about 15 nm to about 40 nm; and, in someembodiments, about 35 nm. The optimization of the dimensions of thepresently disclosed encapsulated SERS-active nanoparticles can beaccomplished by one of ordinary skill in the art. For example, it isknown in the art that core-shell nanoparticles (e.g., Au/AuSnanoparticles) support SERS and have different optical properties ascompared to pure metal nanoparticles. Likewise, it is known in the artthat SERS from prolate spheroids can be enhanced relative to sphereswith the same major axis. Further, it is known that single particleenhancements are wavelength-dependent. Thus, the particle size can be“tuned” to achieve a maximum SERS signal for a given excitationwavelength. Accordingly, the composition of the particle, or its size orshape can be altered in accordance with the presently disclosed subjectmatter to optimize the intensity of the SERS signal.

The presently disclosed composite SERS-active nanoparticles are easy tohandle and store. Further, they also are aggregation resistant,stabilized against decomposition of the dye in solvents and air, arechemically inert, and can be centrifuged, concentrated, e.g., bymagnetic pull down techniques, and redispersed without loss of SERSactivity. Unlike metal nanoparticles, the presently disclosed compositeSERS-active nanoparticles can be evaporated to dryness, and thencompletely redispersed in solvent.

D. Polyethylene Glycol (PEG) Linkers

In some embodiments, a polyethylene glycol (PEG) linker can be used toattach a specific binding member to a SERS-active nanoparticle, amagnetic capture particle (in magnetic capture assays), or to a solidsupport (in heterogeneous assays). The use of a PEG linker can reducenon-specific binding in the presently disclosed assays. Eliminatingnon-specific adsorption can be a significant challenge to assayperformance. For example, in magnetic capture assays, non-specificbinding can include the process in which proteins or other biomoleculesfrom solution adhere to the surfaces of the magnetic capture particle orSERS-active nanoparticle, thereby presenting binding members for thetarget analyte or the process by which the surfaces of the magneticcapture particle and SERS-active nanoparticle adhere to one another vianon-specific interactions.

More generally, non-specific binding refers to binding between moleculesthat is relatively independent of specific surface structures.Non-specific binding can be distinguished from specific binding, whichinvolves the specific recognition of one of two different molecules forthe other compared to substantially less recognition of other molecules.The nature of the molecule or molecules that result in non-specificbinding in liquid-based assays depends on the nature of the sample, theassay milieu, the nanoparticle surface, and the like.

Non-specific binding can be attributed to at least two differentmechanisms, each of which can interfere with an assay. First,non-specific adsorption of biological materials to a SERS-activenanoparticle or a magnetic capture nanoparticle can sterically hinderthe association of the binding molecule with the analyte, resulting in afalse-negative result or an underestimation of the analyte concentrationin a quantitative assay. By “adsorption” is intended the accumulation ofsolutes, biological compounds or other solid materials on the surface ofa solid or the adhesion of a layer of molecules of some substance to thesurface of a solid.

Second, non-specific association between magnetic capture particles andSERS-active nanoparticles, particularly those that have beenfunctionalized, can increase the baseline levels of the assay in theabsence of analyte, leading to a reduction in the sensitivity level ofthe assay and limiting the dynamic range. This baseline increase canresult in a reduction in the minimum level of analyte that can bedetected in a sample. This non-specific association between thenanoparticles, particularly nanoparticles in solution, also can lead tonon-specific aggregation of the particles.

Strategies known in the art for blocking non-specific binding generallyinvolve one of three approaches. In one approach, the surface can betreated with proteins, e.g., albumin, ovalbumin, fish gelatin, andcasein, powdered milk, and/or blocking buffer. Drawbacks to thisapproach include a lack of complete blocking of non-specific adsorption,see, e.g., Taylor, S., et al., “Impact of Surface Chemistry and BlockingStrategies on DNA Microarrays,” Nucleic Acids Research, 31(16), e87(2003), which is incorporated herein by reference in its entirety; theneed to optimize blocking conditions for each new assay; and a varyingperformance from lot to lot of blocking agent. Such blocking steps alsoadd an additional step to the assay, which increases the complexity andduration of the assay.

A second approach involves adding detergents or other chemical agents tothe assay buffer. This approach also suffers from the need to optimizeconditions and reagents for each new assay and can interfere with thespecific biomolecular interactions the assay is intended to detect.

Another approach involves coating the surface of the solid support,nanoparticle, or magnetic particle with a polymer, such as polyethyleneglycol. Without wishing to be bound to any one particular theory, it isthought that the reduction in non-specific adsorption brought about byPEG-coated surfaces is a result of PEG molecules having hydrophilicproperties and having many rotational degrees of freedom. In an aqueousenvironment, the PEG chains are surrounded by water molecules. Theseconditions result in a high level of entropy for the PEG molecules.Adsorption of a biomolecule, e.g., a protein, onto a PEG-coated surfacecompresses the PEG chains, thereby displacing water molecules andimparting order on the PEG chains. This ordering can result in athermodynamically unfavorable drop in entropy resulting in theresistance of biomolecule adsorption on PEG-coated surfaces. Althoughthe coating of two-dimensional surfaces by a polymer, such as PEG, canbe effective in reducing non-specific binding, none of the previouslyutilized approaches are effective in reducing non-specific aggregationof three-dimensional particles in solution. See Dubertret, B., et al.,“In Vivo Imaging of Quantum Dots Encapsulated in Phospholipid Micelles,”Science, 298, 1759-1762 (2002), which is incorporated herein byreference in its entirety.

The presently disclosed subject matter demonstrates that non-specificadsorption of biological materials to the nanoparticle or associationsbetween the nanoparticles, or both, can be reduced through the use ofthe presently disclosed PEG linker (see Experimental Example 3). Thepresently disclosed method is general to blocking non-specific binding.Thus, the need to optimize blocking conditions for individual assays hasbeen largely eliminated. Because polyethylene glycol (PEG)-basedmolecules are non-ionic, the detrimental effect of pH and saltconcentration on the ability of PEG to resist non-specific proteinadsorption is thought to be minimal.

In some embodiments, the PEG linker comprises a bifunctional PEGmolecule having a functional group on either terminal end of the linearmolecule, separated by two or more ethylene glycol subunits. In someembodiments, the PEG molecule comprises between 2 and about 1000ethylene glycol subunits, including but not limited to, 2, at least 3,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9,at least 10, at least 11, at least 12, at least 13, at least 14, atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, at least 21, at least 22, at least 23, at least 24, at least 25, atleast 26, at least 27, at least 28, at least 29, at least 30, at least40, at least 50, at least 60, at least 70, at least 80, at least 90, atleast 100, at least 200, at least 300, at least 400, at least 500, atleast 600, at least 700, at least 800, at least 900, or at least 1000ethylene glycol subunits. In certain embodiments, the PEG moleculecomprises between about to about 100 ethylene glycol subunits, and inparticular embodiments, at least 12 ethylene glycol subunits.

The PEG linker can have a molecular weight of about 200 Da to about100,000 Da, including but not limited to, about 200, about 500, about1,000 Da, about 2,000 Da, about 3,000 Da, about 4,000 Da, about 5,000Da, about 6,000 Da, about 7,000 Da, about 8,000 Da, about 9,000 Da,about 10,000 Da, about 20,000 Da, about 50,000 Da, about 75,000 Da, andabout 100,000 Da. It is to be understood, however, that PEG linkers ofhigher or lower molecular weights can be used with the presentlydisclosed subject matter depending on the particular application. Incertain embodiments, the PEG linker has a molecular weight of about5,000 Da or greater. For a given surface density of PEG molecules,longer PEG chains, e.g., a PEG linker with a molecular weight equal toor greater than about 5,000 Da, can be more effective at reducingnon-specific adsorption in binding assays than shorter chains. LongerPEG chains also can position a binding member, e.g., an antibody or DNAprobe further away from the particle surface. Such embodiments canminimize “folding back” of antibodies onto the particle surface, whichcan reduce the number of available analyte (e.g., antigen) bindingsites.

The bifunctional PEG linker comprises functional groups on each terminalend of the molecule and these functional groups can be used to attach aspecific binding member to one end of the PEG molecule and a SERS-activenanoparticle (or magnetic capture particle) to the other end. Thespecific binding member can first be attached to the bifunctional PEGlinker, followed by attachment of the specific binding member-PEGconjugate to the nanoparticle. Alternatively, the bifunctional PEGlinker can be attached to the nanoparticle prior to attachment of thespecific binding member to the PEGylated nanoparticle.

It is to be noted that a PEG linker also can be used to attach aSERS-active dye to the nanoparticle surface or to attach a specificbinding member to a magnetic capture particle, such as a magneticparticle used in a magnetic capture liquid-based SERS assay.

The functional groups on each terminal end of the PEG linkers can beselected based on the nanoparticle surface chemistry and the desiredfunctionality for attachment of the specific binding member (orSERS-active dye). Non-limiting examples of useful functional groups onPEG linkers include active esters of carboxylic acid or carbonatederivatives, particularly those in which the leaving groups areN-hydroxysuccinimide, p-nitrophenol, imidazole or1-hydroxy-2-nitrobenzene-4-sulfonate. Thiol-reactive groups, includingmaleimido or haloacetyl groups are useful for the modification of freesulfhydryl groups on proteins or for reacting with thiol groups, such asthose that might be present on the surface of SERS-active nanoparticles.

In addition, amino hydrazine or hydrazide groups on PEG molecules areuseful for reaction with aldehydes generated by periodate oxidation ofcarbohydrate groups. This attachment chemistry is particularly usefulfor the site-directed attachment of antibodies onto a particle. Forexample, a bifunctional PEG linker comprising a hydrazine or hydrazidegroup at one terminal end can be used to attach an antibody to thesurface of a SERS-active nanoparticle through the oligosaccharidemoieties on the antibody, which are primarily present in the Fc portionof IgG molecules. In this way, the functionalized nanoparticle can bedesigned to maximize the presentation of the antigen binding region tothe test solution, thereby potentially increasing the sensitivity of theassay. In fact, site-directed immobilization of IgG molecules via theoligosaccharide moieties to hydrazide-derivatized solid supports hasbeen demonstrated to enhance antigenic affinity by about three timesover those IgG molecules immobilized through other types of attachmentchemistries, such as via IgG lysine residues, which are presentthroughout the antibody molecule. See O'Shannessy, D. J. and Hoffman, W.L., Biotechnol. Appl. Biochem. 9, 488-496 (1987); Hoffman, W. L. andO'Shannessy, D. J., J. Immunol. Method, 112, 113-120 (1988).

The bifunctional PEG linker molecule can be homobifunctional orheterobifunctional. As used herein, the term “homobifunctional” refersto a PEG linker molecule in which the terminal functional groups are thesame. The term “heterobifunctional” refers to a PEG linker molecule inwhich the terminal functional groups are different from each other.Thus, in some embodiments, the PEG linker molecule comprises aheterobifunctional PEG molecule comprising a first functional group at afirst terminal end and a second functional group at a second terminalend of the PEG molecule. In some of these embodiments, the firstfunctional group at the first terminal end of the heterobifunctional PEGmolecule comprises a N-hydroxysuccinimide (NHS)-ester. In certainembodiments, the second functional group at the second terminal end ofthe heterobifunctional PEG molecule comprises a maleimide group. Thus,in particular embodiments, the heterobifunctional PEG molecule comprisesa NHS-ester at the first terminal end and a maleimide group at thesecond terminal end.

In those embodiments in which the specific binding member comprises apolynucleotide (e.g., an oligonucleotide), the polynucleotide can beattached to the PEG molecule via an amine group at the 5′-terminal or3′-terminal end of the polynucleotide, which can react with a PEGmolecule that comprises an amine-reactive N-hydroxysuccinimide-ester toform an amino ester (peptide) linkage between the polynucleotide and thePEG linker.

In some embodiments, thiol groups on the surface of a SERS-activenanoparticle are reacted with a thiol-reactive maleimide group on thePEG linker to form a carbon-sulfur bond. In some of these embodiments,the PEG linker comprises a heterobifunctional PEG linker comprising anamine-reactive N-hydroxysuccinimide-ester on one terminal end and amaleimide group on the other terminal end.

In some embodiments, a bifunctional PEG linker can be used to attachspecific binding members to the SERS-active nanoparticles or magneticparticles and the remaining functional groups on the surface of thenanoparticle or magnetic particle can be bound by additional PEGmolecules (e.g., monofunctional PEG molecules) to protect these groupsfrom interacting non-specifically with molecules within the test sampleor other particles. One of ordinary skill in the art upon review of thepresently disclosed subject matter would recognize that any PEGarchitecture, including, but not limited to, linear polymers, starpolymers or copolymers involving PEG, could be used for this purpose.

Further, monofunctional PEG molecules can be used to coat nanoparticlesor magnetic particles comprising a specific binding member that has beenattached thereto with other types of immobilization chemistries known inthe art. For example, streptavidin-biotin coupling chemistry can be usedto attach specific binding members to the particle surface, whereas PEGmolecules, e.g., maleimide-activated PEG molecules, can be used to blocknon-specific adsorption on a thiolated SERS particle surface.

II. Applications of the Presently Disclosed SERS-Active Nanoparticles

In some embodiments, a SERS-active nanoparticle comprising the presentlydisclosed SERS-active reporter molecules can be used in a diagnosticassay for determining the presence or amount of an analyte or ligand ofinterest in a biological sample. Representative diagnostic assays andmethods in which the presently disclosed SERS-active nanoparticles areapplicable are disclosed in PCT International Patent Application No.PCT/US2008/057700 to Weidemaier et al., filed Mar. 20, 2008, which isincorporated herein by reference in its entirety. Accordingly, in someembodiments, the presently disclosed subject matter provides assaymethods, compositions, and kits including SERS-active nanoparticlescomprising the presently disclosed SERS-active reporter molecules.

In some embodiments, a SERS-active nanoparticle comprising the presentlydisclosed SERS-active reporter molecules can be used to detect one ormore of a nucleic acid, e.g., deoxyribonucleic acid (DNA), a DNAfragment, a nucleotide, a polynucleotide, an oligonucleotide, and thelike. Generally, the method comprises contacting one or more of anucleic acid, a DNA fragment, a nucleotide, a polynucleotide, anoligonucleotide, with a presently disclosed SERS-active nanoparticlehaving an oligonucleotide attached thereto and detecting the presence ofor a change in the SERS spectrum thereof.

Further, in some embodiments, a SERS-active nanoparticle comprising thepresently disclosed reporter molecules can be used in cellular imaging.In such embodiments, the presently disclosed SERS-active nanoparticles,for example, a SERS-active nanoparticle labeled with a binding member ofa specific binding pair, can be incorporated into cells or tissues andSERS can be used to characterize the distribution of the nanoparticlestherein. Such embodiments can be used to distinguish between normal andabnormal, e.g., cancerous, cells.

A. Diagnostic Assays

In some embodiments, nanoparticles having one or more presentlydisclosed SERS-active reporter molecules attached thereto can be used indiagnostic assays. For example, Rohr et al. demonstrated an immunoassaywith SERS detection including multiple components and washing steps. SeeRohr, T. E., et al., “Immunoassay employing surface-enhanced Ramanspectroscopy,” Anal. Biochem., 182:388 (1989). Also, Ni et al.demonstrated reporter attachment to a gold slide in a heterogeneousdetection assay including incubation and washing steps. See Ni, J., etal., “Immunoassay Readout Method Using Extrinsic Raman Labels Adsorbedon Immunogold Colloids,” Anal. Chem., 71:4903 (1999). The SERS assaysdisclosed by Rohr et al. and Ni et al., as well as others known in theart, require lengthy incubations and wash steps.

Another example of an assay using SERS is disclosed in U.S. Pat. No.5,266,498 to Tarcha et al., which is incorporated herein by reference inits entirety. Tarcha et al. discloses the use of a multiple reagentsystem in which a label or antibody is attached to a SERS surface. Asecond reagent contains the complementary pair of either label orantibody.

In some embodiments, the presently disclosed SERS-active nanoparticlescan be used in a so-called “liquid-based assay.” Liquid-based assayapproaches using SERS-active nanoparticles have been previouslydisclosed. See, e.g., Hirsch et al., “A Whole Blood Immunoassay UsingGold Nanoshells,” Anal. Chem., 75 (10), 2377-2381 (2003), which isincorporated herein by reference in its entirety. Hirsch et al.discloses the optical detection of particle aggregation in the presenceof an analyte of interest by measuring optical absorption changes due toparticle interactions. The aggregation of the nanoparticles in the assaydisclosed by Hirsch et al. detects the plasmon resonance decrease thatoccurs as a result of the aggregation of particles. Hirsch et al.,however, does not disclose the use of Raman signals for detection.

In one embodiment of the presently disclosed assays, SERS-activeparticles can be used in a so-called “no-wash” or “homogeneous” assay.In such an assay, a sample is collected into a container, e.g., aspecimen collection container, an assay vessel, or other samplecontainer suitable for use with the presently disclosed assays, and theassay is performed without the need to remove sample from the container,e.g., an assay vessel. Advantageously, the sample can be collected intoa container that can already contain all reagents necessary to performthe assay. In some embodiments, however, one or more reagents can beadded to the container following specimen collection.

In liquid-based assays, the sample typically is incubated, e.g., atambient conditions, but it also is possible to provide controlledconditions, such as a specific temperature or rocking of the sample.Following the incubation period, the container can then be placed into areader to obtain a signal from one or more SERS-active particles thatwere pre-loaded or subsequently added into the container. A Raman signalis produced, and detected, upon interrogation by incident radiation of aparticular wavelength, e.g., laser radiation.

In other embodiments, the presently disclosed SERS-active nanoparticlescan be used in heterogeneous assays. As used herein, the term“heterogeneous assay” generally refers to an assay in which one or morecomponents of the assay are added or removed from the assaysequentially. More particularly, a heterogeneous assay can rely, inpart, on the transfer of analyte from a liquid sample to a solid phaseby the binding of the analyte during the assay to the surface of thesolid phase. At some stage of the assay, whose sequence varies dependingon the assay protocol, the solid phase and the liquid phase areseparated and the determination leading to detection and/or quantitationof the analyte is performed on one of the two separated phases. Thus, aheterogeneous assay, for example, can include a solid support coatedwith an antigen or antibody that binds an analyte of interest andthereby separates or removes the analyte from other components in thesample under test. These other components can be selectively removedfrom the sample by one or more washing steps and the analyte remainsbound to the solid support, where it is detected, or can be removed byan additional washing step and subsequently detected.

Generally, the presently disclosed SERS-active nanoparticles can be usedin immunoassays when conjugated to an antibody against a target moleculeof interest. In some embodiments, the presently disclosed dyes can beattached, e.g., covalently attached to a nanoparticle, and used in adiagnostic assay where the intensity of the SERS signal arising from thedye changes as a function of the amount of analyte, e.g., proteins,nucleic acids, and metabolites, detected. Further, in some embodiments,the nanoparticles labeled with the presently disclosed dyes also can belabeled with another species, such as a specific member of a bindingpair, for example, an antibody, to facilitate the detection of one ormore analytes in a sample under test. Nanoparticles having the presentlydisclosed dyes associated with or attached thereto can be used inassays, for example, biological or chemical assays, in which adetectable label is required.

In some embodiments, the presently disclosed subject matter provides amethod for detecting the presence or amount of one or more analytes in abiological sample, the method comprising:

(a) providing a biological sample suspected of containing one or moreanalytes;

(b) contacting the biological sample with a reagent comprising one ormore SERS-active nanoparticles having associated therewith at least onespecific binding member having an affinity for the one or more analytesand at least one SERS-active reporter molecule of Formula:

A-Y

wherein:

A is selected from the group consisting of:

wherein X₁ is CR₄ or N;

Y is selected from the group consisting of:

-   -   wherein:

r, s, and t are each independently an integer from 1 to 8;

each X₂ and X₃ is independently selected from the group consisting of C,S, and N, under the proviso that (i) when X₂ is C or S, R₅ is Z, or whenX₃ is C or S, R₆ is Z, as Z is defined herein below; (ii) if both X₂ andX₃ are N at the same time, at least one of R₅ and R₆ is absent; and(iii) when X₂ is N, R₅ when present is Z′, or when X₃ is N, R₆ whenpresent is Z′, wherein Z′ is selected from the group consisting of:

—(CH₂)_(n)—X₄; —NR₈—(CH₂)_(p)—X₅; —(CH₂)_(q)X₆C(═O)—R₉,

wherein:

n, p, q, u, and v are each independently an integer from 1 to 8;

X₄ and X₅ are each independently selected from the group consisting ofhydroxyl, amino, and thiol;

X₆ is O or NR₁₁;

wherein:

each R₁, R₂, R₃, R₄, R₅, R₆, R₈, R₁₀, R₁₁, and Z is independentlyselected from the group consisting of H, alkyl, substituted alkyl,heteroalkyl, substituted heteroalkyl, cycloalkyl, substitutedcycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl,substituted aryl, aralkyl, hydroxyl, alkoxyl, hydroxyalkyl,hydroxycycloalkyl, alkoxycycloalkyl, aminoalkyl, acyloxyl,alkylaminoalkyl, and alkoxycarbonyl;

R₇ is Z′;

R₉ is —(CH₂)_(m)—X₇ or —(CH₂)_(m)—B, wherein

m is an integer from 1 to 8;

X₇ is halogen; and

B is a binding member having a binding affinity for a ligand or analyteto be detected;

(c) illuminating the biological sample with incident radiation at awavelength to induce the SERS-active reporter molecule to produce a SERSsignal; and

(d) measuring the SERS signal to detect the presence or amount of one ormore analytes in the biological sample.

In some embodiments, the method further comprises continuously: (a)contacting the binding member with the sample suspected of containingone or more analytes; (b) irradiating the sample with electromagneticradiation; and (c) detecting the SERS signal. Accordingly, the detectioncan be carried out continuously or intermittently at predeterminedtimes. Thus, episodic or continuous sensing of analyte(s) of interestcan be performed.

Surface-Immobilized Target Analyte(s) of Interest

In some embodiments, the presently disclosed SERS-active nanoparticlescan be used as optical tags in biological assays. In certain assays, atarget molecule, e.g., an antigen, to be detected is captured by a solidsurface. A binding partner, such as a ligand, e.g., an antibody,specific to the target molecule can be attached to a SERS-activenanoparticle. When contacted with the solid surface having the targetmolecule attached thereto, the SERS-active nanoparticle having thespecific binding partner attached thereto can bind to the targetmolecule. The observation of a SERS signal at the solid supportindicates the presence of the target molecule. Generally, the presentlydisclosed SERS-active nanoparticles can be conjugated to any moleculethat can be used to detect the presence of a specific target molecule inan assay.

More particularly, the target analyte(s) of interest can be immobilized,for example, on a localized area of a solid support, such as afunctionalized inner surface of an assay vessel, e.g., a specimencollection container. Alternatively, in a sandwich assay, the targetanalyte(s) of interest can be immobilized on a solid support indirectlythrough the binding of the analyte to a specific binding member that hasbeen immobilized on the solid support. The immobilized target analyte(s)of interest can then be contacted with a detection reagent comprisingSERS-active nanoparticles conjugated with at least one specific bindingmember, e.g., an antibody, having an affinity for the target analyte(s)of interest. In the sandwich assay, the immobilized specific bindingmember interacts with a separate surface, site, or sequence on theanalyte of interest than the specific binding member that is attached tothe SERS-active nanoparticle, resulting in the analyte being sandwichedbetween the solid support and the nanoparticle, thus generating adetectable SERS signal. In some of these embodiments, the specificbinding member can be immobilized to the solid support or to theSERS-active nanoparticle through a linker, e.g., polyethylene glycol(PEG).

The SERS-active nanoparticles can interact or associate with, e.g., bereversibly or irreversibly bound to, the immobilized target analyte(s)of interest. Following a suitable incubation time, this interactionbetween the SERS-active nanoparticle and the immobilized targetanalyte(s) can be detected by illuminating the localized area of thesolid support with incident radiation of the appropriate wavelength andmeasuring the SERS signal emitted by the SERS-active reporter molecule.Further, because each type of SERS-active reporter molecule exhibits aunique SERS spectrum, a single SERS spectrum can be used to detect aplurality of target analytes of interest by including SERS-activenanoparticles comprising different SERS-active reporter molecules in thedetection reagent. Accordingly, the presently disclosed SERS-activenanoparticles can be used in multiplexed assay formats.

2. Surface-Immobilized Functionalized SERS-Active Nanoparticles

In some embodiments, the presently disclosed SERS-active nanoparticles,conjugated with a specific binding member having an affinity for thetarget analyte(s) of interest, can be immobilized on a localized area ofa solid surface, for example, a functionalized inner surface of aspecimen collection container. The immobilized SERS-active nanoparticlescan be contacted with a biological sample, e.g., a blood sample,suspected of containing one or more target analyte(s) of interest. Theimmobilized SERS-active nanoparticles can interact or associate with,e.g., be reversibly or irreversibly bound to, the target analyte(s) ofinterest present in the sample. Following a suitable incubation time,this interaction between the immobilized SERS-active nanoparticle andthe target analyte(s) can be detected by illuminating the localized areaof the solid support with incident radiation of the proper wavelengthand measuring the SERS signal emitted by the SERS-active reportermolecule. Further, because each type of SERS-active reporter moleculeexhibits a unique SERS spectrum, a single SERS spectrum can be used todetect a plurality of target analytes of interest by immobilizingSERS-active nanoparticles comprising different SERS-active reportermolecules on one or more localized areas of the solid surface. The oneor more localized areas of the solid surface can be illuminated withincident radiation of the appropriate wavelength and the SERS signalemitted by the SERS-active reporter molecule(s) can be measured toprovide a multiplexed diagnostic assay.

3. Liquid-Based SERS Assays

In some embodiments, the presently disclosed SERS-active nanoparticlescan be used in a liquid-based assay. In such assays, nanoparticles canbe prepared according to known methods in the art. The nanoparticles canbe solid nanoparticles, hollow nanoparticles, or encapsulatednanoparticles comprising a solid or hollow nanoparticle core and anencapsulating shell, as disclosed herein. The presently disclosedSERS-active reporter molecules can be adsorbed onto or attached, e.g.,covalently attached through a chemical linker, e.g., a polyethyleneglycol (PEG) linker, to the outer surface of the nanoparticle or thenanoparticle core. Binding members of a specific binding pair can beattached to an outer surface of the nanoparticle, conjugated to thereporter molecule adsorbed on or attached to the outer surface of thenanoparticle, or attached to an outer surface of the encapsulatingshell. In some embodiments of the presently disclosed assays, thebinding member is an antibody. The SERS-active nanoparticle having abinding member of a specific binding pair attached thereto can then becontacted with a biological sample suspected of containing one or moretarget analytes or ligands of interest. The specific binding member canassociate, e.g., bind with, the one or more target analytes or ligandsof interest.

In some liquid-based assays, a sandwich assay can be used to amplify theSERS signal as disclosed in PCT International Patent Application No.PCT/US2005/000171 to Wang et al., filed Jan. 6, 2005, which isincorporated herein by reference in its entirety. In such assays, aparticular analyte is able to simultaneously bind through multiplesurfaces, sites, or sequences on or within the analyte to more than onebinding member, wherein the more than one binding member is attached toSERS-active nanoparticles. In those instances wherein the SERS-activenanoparticles that are simultaneously bound to the analyte through morethan one binding member have attached thereto identical SERS-active dyesor SERS-active dyes exhibiting overlapping SERS spectra, the SERS signalcan be amplified. It is believed that this sandwich structure orclustering of SERS-active nanoparticles around an analyte moleculegenerates an amplified SERS effect through an increase in the effectivesurface size of the clustered metallic particles, an enhancement in theelectromagnetic field at the midpoint between closely spaced metallicparticles, and additional local field enhancement mechanisms arisingfrom sharp edges, kinks, or other fractal structures provided by thecluster structure.

Further, the presently disclosed dyes exhibit relatively simple Ramanspectra with narrow line widths. This characteristic allows for thedetection of several different Raman-active species in the same samplevolume. Accordingly, this feature allows multiple SERS-activenanoparticles, each including different dyes, to be fabricated such thatthe Raman spectrum of each dye can be distinguished in a mixture ofdifferent types of nanoparticles. This feature allows for the multiplexdetection of several different target species in a small sample volume.Thus, nanoparticles having the presently disclosed dyes associated withor attached thereto also are suitable for use in multiplexed chemicalassays, in which the identity of the SERS-active nanoparticle encodesthe identity of the target of the assay.

Accordingly, in some embodiments, more than one type of binding membercan be attached to the nanoparticle. For example, the type of bindingmember attached to the nanoparticle can be varied to provide multiplereagents having different affinities for different target analytes. Inthis way, the assay can detect more than one analyte of interest orexhibit different selectivities or sensitivities for more than oneanalyte. The SERS-active nanoparticle can be tailored for samples inwhich the presence of one or more analytes, or the concentrations of theone or more analytes, can vary.

The presently disclosed dyes of Formula A-Y, when associated with orattached to SERS-active nanoparticles, provide spectral diversity andresolvability in multiplex assays. Each SERS-active nanoparticle, whencoupled to a target-specific reagent, can encode the identity of thatparticular target molecule. Further, the intensity of a particular Ramansignal can reveal the quantity of that particular target molecule. Forexample, as described hereinabove for sandwich assays, the identity ofdifferent targets captured on a solid support can be determined by usingfor each target SERS-active nanoparticles having different dyes ofFormula A-Y associated with or attached thereto. Accordingly, thepresently disclosed SERS-active nanoparticles can be used in multiplexedassays to yield qualitative and/or quantitative information regarding atarget molecule without requiring position-sensitive localization ofreagents.

A liquid-based SERS assay reagent can include more than one type oflabel, e.g., more than one type of SERS-active reporter molecule,depending on the requirements of the assay. For example, SERS-activereporter molecules exhibiting a Raman signal at different wavelengthscan be used to create a unique Raman “fingerprint” for a specificanalyte of interest, thereby enhancing the specificity of the assay.Different reporter molecules can be attached to different specificbinding members to provide a single detection reagent capable ofdetecting more than one analyte of interest, e.g., a plurality ofanalytes of interest. Further, multiple reporter molecules can be usedto create an internal reference signal that can be used to distinguishbackground noise from signal detection, particularly in samples thatexhibit or are expected to exhibit a relatively weak signal.Additionally, more than one SERS-reporter molecule can be used to avoidor overcome non-specific radiation emitted from the sample solutionunder test, i.e., radiation emitted from the sample solution that cannotbe attributed to direct or indirect measurement of an analyte ofinterest.

Further, methods for amplifying a SERS signal in a liquid-based assay,as disclosed in PCT International Patent Application No.PCT/US2008/057700 to Weidemaier et al., filed Mar. 20, 2008, which isincorporated herein by reference in its entirety, also are applicablefor use with the presently disclosed SERS-active nanoparticles.

4. Magnetic Capture Liquid-Based SERS Assays

In some embodiments, the presently disclosed liquid-based SERS assayreagents can be used in a magnetic capture assay. In such embodiments,the components of the assay, including the particles, labels, andspecific binding members, are introduced to a sample under testsuspected of containing one or more analytes of interest. Upon allowingthe reagent to interact with the complex solution, the nanoparticles canbe localized using the magnetic properties of the particles. In someembodiments, a magnetic capture reagent can be used to facilitatelocalization of the SERS-active nanoparticles. In such embodiments,magnetic particles can be labeled with a binding member that has anaffinity for one or more analytes of interest. The magnetic propertiesof the magnetic particles can be used to localize the SERS-activenanoparticle-analyte complex for detecting the SERS signal.Representative methods for conducting magnetic capture liquid-based SERSassays are disclosed in PCT International Patent Application No.PCT/US2008/057700 to Weidemaier et al., filed Mar. 20, 2008, which isincorporated herein by reference in its entirety. Such methods caninclude referencing and control methods for compensating for variationsin magnetic pellet size, shape, or positioning, and methods forgenerating improved Raman reference spectra and spectral analysis inmagnetic pull-down liquid-based assays, as also disclosed inPCT/US2008/057700.

In some embodiments, the SERS-active nanoparticle-analyte complex islocalized at a predetermined area within the sample container, forexample, a sample collection tube. Radiation can then be directed at thelocalization area and the signal can be detected. The localization ofthe single detection reagent can increase the reporter molecule-surfaceinteraction and increase the signal by concentrating the SERS effect toa particular area of the sample container.

Magnetic capture of the particles can be accomplished using any methodknown in the art, including, but not limited to, placing a strong magnetor inducing a magnetic field at a localized area of the samplecollection container.

More particularly, in some embodiments, the presently disclosedSERS-active nanoparticles, conjugated with a specific binding memberhaving an affinity for the target analyte(s) of interest can be disposedin a specimen collection container either prior to, concurrent with, orsubsequent to disposing therein a biological sample suspected ofcontaining one or more target analytes of interest. Magnetic particles,also conjugated with a specific binding member having an affinity forthe target analyte(s) of interest, can be disposed in the specimencollection container. Target analyte(s) of interest present in thesample can bind to the SERS-active nanoparticles and the magneticparticles, thereby forming a complex wherein the target analyte(s) issandwiched between the SERS-active nanoparticle and the magneticparticle. The sandwich complexes can be concentrated in a localized areaof the sample collection container by application of a magnetic field.Following a suitable incubation time, the sandwich complexes can bedetected by illuminating the localized area of the sample collectioncontainer with incident radiation of the appropriate wavelength andmeasuring the SERS signal emitted by the SERS-active reporter molecule.

As described herein, a PEG linker can attach the specific binding memberto the SERS-active nanoparticle surface to reduce non-specific bindingof molecules to the nanoparticle. In some embodiments wherein a magneticcapture assay is performed, a specific binding member can be attached tothe surface of a magnetic particle through a linker. In some of theseembodiments, the linker molecule comprises a PEG linker. The PEG linkercan vary in length, molecular weight, and functional groups useful forlinking the specific binding member to the magnetic particle, asdescribed herein.

In some embodiments, the PEG molecule or PEG-specific binding memberconjugate is attached to the magnetic particle through the reaction ofthiol-reactive maleimide groups on the PEG molecule with thiol groups onthe magnetic particle surface to form a carbon-sulfur bond. The surfaceof the magnetic particle can be functionalized with a thiol group bytreating a carboxylated magnetic particle with an amine-terminatedmolecule containing an internal disulfide. The disulfide can be cleavedwith dithiothreitol or other suitable agent, exposing a reactive thiolgroup. In some of these embodiments, the PEG linker comprises aheterobifunctional PEG linker with an amine-reactiveN-hydroxysuccinimide-ester on one end and a maleimide group on the otherend.

5. Representative Target Analytes of Interest

The presently disclosed methods can be used to assess or measure thepresence or amount of one or more target analytes in a biologicalsample. The term “analyte,” as used herein, generally refers to asubstance to be detected, which can be present or suspected of beingpresent in a test sample. More particularly, an “analyte” can be anysubstance for which there exists a naturally occurring specific binderpartner, such as a binding protein or receptor, or for which a specificbinding partner can be prepared. Accordingly, an “analyte” is asubstance that can bind one or more specific binding partners in anassay. In some embodiments, the analyte can be any compound, such as ametabolite, to be detected or measured and which has at least onebinding site.

The target analytes can be any molecule or compound, of which thepresence or amount is to be determined in a sample under test. Examplesof classes of analytes that can be measured by the presently disclosedmethods include, but are not limited to amino acids, peptides,polypeptides, proteins, carbohydrates, fatty acid, lipids, nucleotides,oligonucleotides, polynucleotides, glycoproteins, such as prostatespecific antigen (PSA), proteoglycans, lipoproteins,lipopolysaccharides, drugs, drug metabolites, small organic molecules,inorganic molecules and natural or synthetic polymers. Examples oftarget analytes include, but are not limited to, glucose, free fattyacids, lactic acid, C-reactive protein and anti-inflammatory mediators,such as cytokines, eicosanoids, or leukotrienes. In some embodiments,the target analytes are selected from the group consisting of fattyacids, C-reactive protein, and leukotrienes. In another embodiment, thetarget analytes are selected from the group consisting of glucose,lactic acid and fatty acids.

More particularly, in some embodiments, the analyte can include glucose,as described hereinabove, prostate specific antigen (PSA), creatinekinase MB (CKMB) isoenzyme, cardiac troponin I (cTnI) protein,thyroid-stimulating hormone (TSH), influenza A (Flu A) antigen,influenza B (Flu B) antigen, and respiratory syncytial virus (RSV)antigen.

Prostate specific antigen (PSA) is a protein produced by the cells ofthe prostate gland and typically is present in small quantities in theserum of normal men. PSA can be elevated in men afflicted with prostatecancer or other prostate disorders. Normal PSA blood levels typicallyare considered to be between about 0.0 and 4.0 ng/mL, whereas PSA levelsbetween 4 and 10 ng/mL (nanograms per milliliter) are consideredsuspicious.

Creatine kinase (CK), also known as phosphocreatine kinase or creatinephosphokinase (CPK) is an enzyme found predominately in the heart,brain, and skeletal muscle. Creatine kinase comprises three isoenzymesthat differ slightly in structure: CK-BB (also referred to as CPK-1) isconcentrated in the brain and lungs; CK-MB (also referred to as CPK-2)is found mostly in the heart; and CK-MM (also referred to as CPK-3) isfound mostly in skeletal muscle. Diagnostic tests for specific CPKisoenzymes typically are performed when the total CPK level is elevatedand can help differentiate the source of the damaged tissue. Forexample, an injury to the brain, e.g., a stroke, or lungs, e.g., apulmonary embolism, can be associated with elevated levels of CK-BB.Further, CK-MM is normally responsible for almost all CPK enzymeactivity in healthy subjects. When this particular isoenzyme iselevated, it usually indicates injury or stress to skeletal muscle.

CK-MB levels can be measured in subjects who have chest pain to diagnosewhether they had a heart attack and/or as an as an indication formyocardial damage during heart attacks. Typically, CK-MB values exhibita significant rise in CK-MB values in the first two to three hours aftera heart attack. If there is no further damage to the heart muscle, thelevel peaks at 12-24 hours and returns to normal 12-48 hours aftertissue death. CK-MB levels do not usually rise with chest pain caused byangina, pulmonary embolism (blood clot in the lung), or congestive heartfailure. Elevated CK-MB levels also can be observed in subjectssuffering from myocarditis (inflammation of the heart muscle, forexample, due to a virus), electrical injuries, trauma to the heart,heart defibrillation, and open heart surgery. Blood serum CK-MB valuesmeasured in such assays typically range from about 0.0 to about 10ng/mL. CK-MB values greater than about 5 ng/mL typically confirm adiagnosis of myocardial infarction.

Cardiac troponin I (cTnI) protein also is an independent predictor ofmajor cardiac events. See, e.g., Polancyzk, C. A., et al., “Cardiactroponin I as a predictor of major cardiac events in emergencydepartment patients with acute chest pain,” J. Am. Coll. Cardiol., 32,8-14 (1998). cTnI values in blood serum measured in subject suspected ofhaving a myocardial infarction range from about 0.4 ng/mL to about 1.5ng/mL. Id. cTnI assays with lower detection limits of 0.1 ng/mL have thepotential, however, to be more sensitive for detecting myocardialinjury. Id.

Thyroid-stimulating hormone (TSH) is synthesized and secreted bythyrotrope cells in the anterior pituitary gland which regulates theendocrine function of the thyroid gland. TSH levels are tested in theblood of subjects suspected of suffering from an excess(hyperthyroidism) or deficiency (hypothyroidism) of thyroid hormone.Normal TSH levels in adults range from about 0.4 milli-internationalunits per liter (mIU/L) to about 4.5 mIU/L. Current assays for TSHinclude sandwich ELISA for the measurement of TSH in blood serum orplasma, in which TSH in the sample is bound by anti-TSH monoclonalantibodies and then detected by spectrophotometry or colorimetry.

The presently disclosed assays also can be used to detect influenzaviruses. Three types of influenza viruses exist: Influenzavirus A;Influenzavirus B; and Influenzavirus C. Influenza A (Flu A) andInfluenza C (Flu C) infect multiple species, while Influenza B (Flu B)infects almost exclusively humans. Type A viruses are the most virulenthuman pathogens among the three influenza types and typically cause themost severe disease. Influenza A virus can be subdivided into differentserotypes based on the antibody response to these viruses and includeH1N1 (i.e., “Spanish Flu”); H2N2 (i.e., “Hong Kong Flu”); H5N1 (i.e.,avian influenza strain or “Bird Flu”); H7N7; H1N2; H9N2; H7N2; H7N3, andH10N7. Influenza B is almost exclusively a human pathogen and is lesscommon than Influenza A and only includes one serotype. The influenza Cvirus infects humans and pigs and can cause severe illness and localepidemics, but is less common than the other types.

Diagnostic tests available for influenza include rapid immunoassay,immunofluorescence assay, polymerase chain reaction (PCR), serology, andviral culture. Immunofluorescence assays entail staining of specimensimmobilized on microscope slides using fluorescent-labeled antibodiesfor observation by fluorescence microscopy. Culture methods employinitial viral isolation in cell culture, followed by hemadsorptioninhibition, immunofluorescence, or neutralization assays to confirm thepresence of the influenza virus. Antigen detection assays to diagnoseinfluenza infection include DIRECTIGEN™ EZ Flu A or DIRECTIGEN™ EZ FluA+B test kits, (available from BD Diagnostic Systems, Sparks, Md.). Suchrapid chromatographic immunoassays can be used for the direct detectionof influenza A or influenza A and B viral antigens from nasopharyngealwashes/aspirates, nasopharyngeal swabs and throat swabs of symptomaticpatients. Further, such diagnostic tests can be used to distinguishbetween influenza A and influenza B.

Respiratory syncytial virus (RSV) is the most common cause ofbronchiolitis and pneumonia among infants and children under 1 year ofage. RSV is a negative-sense, enveloped RNA virus. Diagnosis of RSVinfection can be made by virus isolation, detection of viral antigens,detection of viral RNA, demonstration of a rise in serum antibodies, ora combination of these approaches. Traditional methods for detection ofrespiratory viruses have included cell culture and direct fluorescentantibody (DFA). Enzyme immunoassay (EIA) and rapid manual systems areavailable for specific viruses such as Influenza A/B and RSV. Currently,most clinical laboratories use antigen detection assays to diagnose RSVinfection, such as DIRECTIGEN™ EZ RSV test (available from BD DiagnosticSystems, Sparks, Md.), which is a rapid chromatographic immunoassay forthe direct and qualitative detection of RSV antigen in nasopharyngealwashes, nasopharyngeal aspirates, nasopharyngeal swabs andnasopharyngeal swab/washes from subjects suspected of having a viralrespiratory infection.

Accordingly, in some embodiments, the presently disclosed subject matterprovides a method for detecting the presence or amount of a targetanalyte in a biological sample, e.g., blood serum, wherein the targetanalyte includes glucose, prostate specific antigen (PSA), creatinekinase MB (CKMB) isoenzyme, cardiac troponin I (cTnI) protein,thyroid-stimulating hormone (TSH), influenza A (Flu A) antigen,influenza B (Flu B) antigen, and respiratory syncytial virus (RSV)antigen, the method comprising contacting the biological sample with areagent comprising one or more SERS-active nanoparticles havingassociated therewith at least one specific binding member having anaffinity for the analyte, e.g., a specific binding protein or monoclonalor polyclonal antibody for the analyte of interest, and at least oneSERS-active reporter molecule of Formula A-Y; illuminating thebiological sample with incident radiation at a wavelength to induce theSERS-active reporter molecule to produce a SERS signal; and measuringthe SERS signal to detect the presence or amount of analyte in thebiological sample.

As used herein, the term “carbohydrate” includes, but is not limited tomonosaccharides, disaccharides, oligosaccharides and polysaccharides.“Carbohydrate” also includes, but is not limited to, moleculescomprising carbon, hydrogen and oxygen that do not fall within thetraditional definition of a saccharide, i.e., an aldehyde or ketonederivative of a straight chain polyhydroxyl alcohol, containing at leastthree carbon atoms. Thus, for example, a carbohydrate as used herein cancontain fewer than three carbon atoms.

The term “fatty acids,” as used herein include all fatty acids,including free fatty acids (FFA) and fatty acids esterified to othermolecules. Examples of specific fatty acids include, but are not limitedto, palmitate, stearate, oleate, linoleate, linolenate, andarachidonate. The term “free fatty acid” is used herein as it is knownin the art in that FFA are not part of other molecules, such astriglycerides or phospholipids. Free fatty acids also includenon-esterified fatty acids that are bound to or adsorbed onto albumin.As used herein, the term “unbound free fatty acid” (unbound FFA) is usedto denote a free fatty acid or free fatty acids that are not bound oradsorbed onto albumin or other serum proteins.

As used herein, the term “lipid” is used as it is in the art, i.e., asubstance of biological origin that is made up primarily or exclusivelyof nonpolar chemical groups such that it is readily soluble in mostorganic solvents, but only sparingly soluble in aqueous solvents.Examples of lipids include, but are not limited to, fatty acids,triacylglycerols, glycerophospholipids, sphingolipids, cholesterol,steroids and derivatives thereof. For example, “lipids” include but arenot limited to, the ceramides, which are derivatives of sphingolipidsand derivatives of ceramides, such as sphingomyelins, cerebrosides andgangliosides. “Lipids” also include, but are not limited to, the commonclasses of glycerophospholipds (or phospholipids), such as phosphatidicacid, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine,phosphatidylinositol, phosphatidylglycerol, and the like.

As used herein, a “drug” can be a known drug or a drug candidate, whoseactivity or effects on a particular cell type are not yet known. A “drugmetabolite” is any of the by-products or the breakdown products of adrug that is changed chemically into another compound or compounds. Asused herein, “small organic molecule” includes, but is not limited to,an organic molecule or compound that does not fit precisely into otherclassifications highlighted herein. More particularly, the term “smallorganic molecule” as used herein, refers to organic compounds, whethernaturally-occurring or artificially created (e.g., via chemicalsynthesis) that have relatively low molecular weight and that are notproteins, polypeptides, or nucleic acids. Typically, small moleculeshave a molecular weight of less than about 1500 g/mol. Also, smallmolecules typically have multiple carbon-carbon bonds.

Further, in some embodiments, the presently disclosed subject matterprovides a method of detecting one or more of a nucleic acid, e.g.,deoxyribonucleic acid (DNA), a DNA fragment, a nucleotide, apolynucleotide, an oligonucleotide, and the like. Generally, the methodcomprises contacting one or more of a nucleic acid, a DNA fragment, anucleotide, a polynucleotide, an oligonucleotide, with a presentlydisclosed SERS-active nanoparticle having an oligonucleotide attachedthereto and detecting the presence of or a change in the SERS spectrumthereof. In exemplary embodiments, the oligonucleotides attached to thepresently disclosed SERS active nanoparticles have a sequence, orsequences, complementary to portions of the sequence of the targetnucleic acid, DNA fragment, nucleotide, polynucleotide, oroligonucleotide. A detectable SERS spectrum, and/or a change in the SERSspectrum, can be observed as a result of the hybridization of theoligonucleotide attached to the SERS active nanoparticle and the targetnucleic acid, DNA fragment, nucleotide, polynucleotide, oroligonucleotide.

The presently disclosed SERS-active nanoparticles, the oligonucleotides,or both can be functionalized to attach the oligonucleotides to thenanoparticles. Such methods are known in the art. For example,oligonucleotides functionalized with alkanethiols at the 3′-termini or5′-termini readily attach to nanoparticles, including gold and othermetal nanoparticles. See, e.g., Whitesides, Proceedings of the Robert A.Welch Foundation 39th Conference On Chemical Research NanophaseChemistry, Houston, Tex., pp. 109-121 (1996); see also, Mucic et al.,Chem. Commun. 555-557 (1996) (describing a method of attaching 3′ thiolDNA to flat gold surfaces which also can be used to attacholigonucleotides to nanoparticles).

Other functional groups suitable for attaching oligonucleotides to solidsurfaces include phosphorothioate groups (see, e.g., U.S. Pat. No.5,472,881 to Beebe et al., which is incorporated herein by reference inits entirety, for the binding of oligonucleotide-phosphorothioates togold surfaces), substituted alkylsiloxanes (see, e.g., Burwell, ChemicalTechnology, 4, 370-377 (1974) and Matteucci and Caruthers, J. Am. Chem.Soc., 103, 3185-3191 (1981) for binding of oligonucleotides to silicaand glass surfaces, and Grabar et al., Anal. Chem., 67, 735-743 (1995)for binding of aminoalkylsiloxanes and for similar binding ofmercaptoaklylsiloxanes). Oligonucleotides terminated with a5′thionucleoside or a 3′ thionucleoside also can be used for attachingoligonucleotides to solid surfaces.

Other methods are known in the art for attaching oligonucleotides tonanoparticles. Such methods are described in the followingrepresentative references. Nuzzo et al., J. Am. Chem. Soc., 109, 2358(1987) (disulfides on gold); Allara and Nuzzo, Langmuir, 1, 45 (1985)(carboxylic acids on aluminum); Allara and Tompkins, J. ColloidInterface Sci., 49, 410-421 (1974) (carboxylic acids on copper); Iler,The Chemistry Of Silica, Chapter 6, John Wiley & Sons, New York (1979)(carboxylic acids on silica); Timmons and Zisman, J. Phys. Chem., 69,984-990 (1965) (carboxylic acids on platinum); Soriaga and Hubbard, J.Am. Chem. Soc., 104, 3937 (1982) (aromatic ring compounds on platinum);Hubbard, Acc. Chem. Res., 13, 177 (1980) (sulfolanes, sulfoxides andother functionalized solvents on platinum); Hickman et al., J. Am. Chem.Soc., 111, 7271 (1989) (isonitriles on platinum); Maoz and Sagiv,Langmuir, 3, 1045 (1987) (silanes on silica); Maoz and Sagiv, Langmuir,3, 1034 (1987) (silanes on silica); Wasserman et al., Langmuir, 5, 1074(1989) (silanes on silica); Eltekova and Eltekov, Langmuir, 3, 951(1987) (aromatic carboxylic acids, aldehydes, alcohols and methoxygroups on titanium dioxide and silica); Lec et al., J. Phys. Chem., 92,2597 (1988) (rigid phosphates on metals).

Further, oligonucleotides functionalized with a cyclic disulfide, forexample, cyclic disulfides having a 5- to 6-membered ring including atleast two sulfur atoms, also are suitable for use with the presentlydisclosed subject matter. Suitable cyclic disulfides are availablecommercially or can be synthesized by known procedures. The reduced formof the cyclic disulfides also can be used. In some embodiments, thecyclic disulfide can further have a linker, for example, a hydrocarbonmoiety, such as a steroid residue, attached thereto.

In some embodiments, polynucleotides (e.g., oligonucleotides) areattached to the outer surface of a SERS-active nanoparticle through alinker molecule. In particular embodiments, the linker moleculecomprises a PEG linker. The PEG linker can be attached to thepolynucleotide and the nanoparticle through any suitable method,including those described elsewhere herein.

Each nanoparticle can have a plurality of oligonucleotides attachedthereto. As a result, each nanoparticle-oligonucleotide conjugate canbind to a plurality of oligonucleotides or nucleic acids having acomplementary sequence. Methods of making oligonucleotides of apredetermined sequence are well-known. See, e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual (2nd ed. 1989) and F. Eckstein(ed.) Oligonucleotides and Analogues, 1st Ed. (Oxford University Press,New York, 1991). Solid-phase synthesis methods can be used foroligoribonucleotides and oligodeoxyribonucleotides (known methods ofsynthesizing DNA also are useful for synthesizing RNA).Oligoribonucleotides and oligodeoxyribonucleotides also can be preparedenzymatically.

Accordingly, the presently disclosed subject matter provides a methodfor detecting nucleic acids. Any type of nucleic acid can be detected bythe presently disclosed method. Therefore, the presently disclosedmethods can be used in several applications where the detection of anucleic acid is required, for example, in the diagnosis of disease andin sequencing of nucleic acids. Examples of nucleic acids that can bedetected by the presently disclosed methods include, but are not limitedto, genes (e.g., a gene associated with a particular disease), viral RNAand DNA, bacterial DNA, fungal DNA, cDNA, mRNA, RNA and DNA fragments,oligonucleotides, synthetic oligonucleotides, modified oligonucleotides,single-stranded and double-stranded nucleic acids, natural and syntheticnucleic acids, and the like.

Representative examples of the uses of the methods of detecting nucleicacids include, but are not limited to, the diagnosis and/or monitoringof viral diseases (e.g., human immunodeficiency virus, hepatitisviruses, herpes viruses, cytomegalovirus, and Epstein-Barr virus),bacterial diseases (e.g., tuberculosis, Lyme disease, H. pylori,Escherichia coli infections, Legionella infections, Mycoplasmainfections, Salmonella infections), sexually transmitted diseases (e.g.,gonorrhea), inherited disorders (e.g., cystic fibrosis, Duchene musculardystrophy, phenylketonuria, sickle cell anemia), and cancers (e.g.,genes associated with the development of cancer); in forensics; in DNAsequencing; for paternity testing; for cell line authentication; formonitoring gene therapy; and for many other purposes.

The nucleic acid to be detected can be isolated by known methods, or canbe detected directly in cells, tissue samples, biological fluids (e.g.,saliva, urine, blood, serum, and the like), solutions containing PCRcomponents, solutions containing large excesses of oligonucleotides orhigh molecular weight DNA, and other samples, as also known in the art.See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nded. 1989) and B. D. Hames and S. J. Higgins, Eds., Gene Probes 1 (IRLPress, New York, 1995). Methods of preparing nucleic acids for detectionwith hybridizing probes also are well known in the art. See, e.g.,Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed. 1989)and B. D. Hames and S. J. Higgins, Eds., Gene Probes 1 (IRL Press, NewYork, 1995). If a nucleic acid is present in small amounts, it can beapplied by methods known in the art, including polymerase chain reaction(PCR) amplification. See, e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual (2nd ed. 1989) and B. D. Hames and S. J. Higgins,Eds., Gene Probes 1 (IRL Press, New York, 1995).

One presently disclosed method for detecting nucleic acid comprisescontacting a nucleic acid with one or more of the presently disclosednanoparticles having oligonucleotides attached thereto. The nucleic acidto be detected can have at least two portions. The lengths of theseportions and the distance(s), if any, between them are chosen so thatwhen the oligonucleotides on the nanoparticles hybridize to the nucleicacid, a detectable SERS signal can be observed. These lengths anddistances can be determined empirically and depend on the type ofparticle used and its size and the type of electrolyte present insolutions used in the assay (as is known in the art, certainelectrolytes affect the conformation of nucleic acids).

Also, when a nucleic acid is to be detected in the presence of othernucleic acids, the portions of the nucleic acid to which theoligonucleotides on the nanoparticles are to bind must be chosen so thatthey contain sufficient unique sequence so that detection of the nucleicacid will be specific. Guidelines for doing so are well known in theart. The contacting of the nanoparticle-oligonucleotide conjugates withthe nucleic acid takes place under conditions effective forhybridization of the oligonucleotides on the nanoparticles with thetarget sequence(s) of the nucleic acid. These hybridization conditionsare well known in the art and can readily be optimized for theparticular system employed. See, e.g., Sambrook et al., MolecularCloning: A Laboratory Manual (2nd ed. 1989). In some embodiments,stringent hybridization conditions are employed.

Representative methods for detecting nucleic acids by using SERS-activenanoparticles having oligonucleotides attached thereto are disclosed inU.S. Pat. No. 7,169,556 to Park et al., which is incorporated herein byreference in its entirety.

6. Representative Instrumentation for Detecting a SERS Signal Emitted bya

Sample Under Test

In some embodiments, a laser serves as the excitation source of theincident radiation used to detect one or more target analytes ofinterest. One of ordinary skill in the art upon review of the presentlydisclosed subject matter could ascertain the type of laser, includingthe strength and excitation wavelength, suitable for use with theSERS-active reporter molecules described herein. Radiation scattered oremitted from the sample can be detected using detection systems known inthe art.

In some embodiments, more than one type of radiation source, or morethan one excitation wavelength, can be used. For example, in embodimentswherein two analytes of interest are to be detected, the singledetection reagent can include two distinct types of SERS-active reportermolecules and/or two distinct types of specific binding members.Accordingly, incident radiation of different wavelengths can be used toproduce distinct Raman signals for each analyte of interest. As one ofordinary skill in the art would recognize upon review of the presentlydisclosed subject matter, the selection of the particular wavelength(s)to be used depends on the analyte of interest, the specific bindingmembers used, and the particular SERS-active reporter molecules used.

The presently disclosed assay can be conducted with any suitable Ramanspectrometer systems known in the art, including, for example, aMultimode Multiple Spectrometer Raman Spectrometer (Centice,Morrisville, N.C., United States of America), such as the Ramanspectrometer system disclosed in U.S. Pat. No. 7,002,679 to Brady etal., which is incorporated herein by reference in its entirety.Additional instrumentation suitable for use with the presently disclosedSERS-active nanoparticles is disclosed in PCT International PatentApplication No. PCT/US2008/057700 to Weidemaier et al., filed Mar. 20,2008, which is incorporated herein by reference in its entirety.

Sensing devices, such as optical detectors, radiation sources, andcomputer systems, microprocessors, and computer software and algorithms,can be used in any combination in practicing the methods disclosedherein. Accordingly, in some embodiments, software, or other computerreadable instructions can be used to interpret, analyze, compile, orotherwise parse output data related to the presently disclosed opticalassay. The software or other computer system can be used to display,store, or transmit output data, whether in digital or other forms to oneor more users.

7. Sample Collection Container

In some embodiments, the sample container is selected from the groupconsisting of a cuvette, a tube, such as a blood collection tube, or anyother sample collection container compatible with the sample under testand SERS measurements. In some embodiments, the sample collectioncontainer, e.g., a tube, can have an internal pressure that is less thanthe atmospheric pressure of the surrounding environment. Such samplecollection containers are disclosed in U.S. Pat. No. 5,860,937 to Cohen;U.S. Pat. No. 5,906,744 to Carroll et al.; and U.S. Pat. No. 6,821,789to Augello et al., each of which is incorporated herein by reference intheir entirety. Additional assay vessels suitable for use with thepresently disclosed SERS-active nanoparticles, in particular for use inmagnetic capture assays, are disclosed in PCT International PatentApplication No. PCT/US2008/057700 to Weidemaier et al., filed Mar. 20,2008, which is incorporated herein by reference in its entirety.Further, in some embodiments, the sample collection container includes asingle detection reagent comprising the presently disclosed SERS-activenanoparticles. In such embodiments, the sample collection container hasa single detection reagent disposed therein before the user, e.g., apatient or a medical technician, collects the biological sample, e.g.,blood, to be detected. The single detection reagent, for example, can beimmobilized on an inner surface, e.g., an inner wall, of the samplecollection container or simply otherwise disposed within the samplecontainer.

The sample collection container, for example, a blood collection tube,can be shipped to the user with the single detection reagent disposedtherein. Alternatively, the user can select a suitable detection reagentand introduce the detection reagent into the collection device beforecollecting the sample specimen. Further, the presently disclosed subjectmatter can include a kit comprising one or more of a sample collectioncontainer, such as a blood collection tube, one or more reagents, suchas one or more single detection reagents comprising nanoparticles havinga SERS-active reporter molecule attached thereto, magnetic captureparticles, and individual components thereof. Such kits can include anynumber of the components of the assay, including, but not limited to,multiple reporter molecules or multiple specific binding members eitherattached to a nanoparticle or packaged separately therefrom.

As used herein, the term “sample” includes any liquid or fluid sample,including a sample derived from a biological source, such as aphysiological fluid, including whole blood or whole blood components,such as red blood cells, white blood cells, platelets, serum and plasma;ascites; urine; saliva; sweat; milk; synovial fluid; peritoneal fluid;amniotic fluid; percerebrospinal fluid; lymph fluid; lung embolism;cerebrospinal fluid; pericardial fluid; cervicovaginal samples; tissueextracts; cell extracts; and other constituents of the body that aresuspected of containing the analyte of interest. In addition tophysiological fluids, other liquid samples, such as water, food productsand the like, for the performance of environmental or food productionassays are suitable for use with the presently disclosed subject matter.A solid material suspected of containing the analyte also can be used asthe test sample. In some instances it might be beneficial to modify asolid test sample to form a liquid medium or to release the analyte.

In some embodiments, the sample can be pre-treated prior to use, such aspreparing plasma from blood, diluting viscous fluids, or the like. Suchmethods of treatment can involve filtration, distillation,concentration, inactivation of interfering compounds, and the additionof reagents.

The sample can be any sample obtained from a subject. The term “subject”refers to an organism, tissue, or cell from which a sample can beobtained. A subject can include a human subject for medical purposes,such as diagnosis and/or treatment of a condition or disease, or ananimal subject for medical, veterinary purposes, or developmentalpurposes. A subject also can include sample material from tissueculture, cell culture, organ replication, stem cell production and thelike. Suitable animal subjects include mammals and avians. The term“avian” as used herein includes, but is not limited to, chickens, ducks,geese, quail, turkeys, and pheasants. The term “mammal” as used hereinincludes, but is not limited to, primates, e.g., humans, monkeys, apes,and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g.,sheep and the like; caprines, e.g., goats and the like; porcines, e.g.,pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, andthe like; felines, including wild and domestic cats; canines, includingdogs; lagomorphs, including rabbits, hares, and the like; and rodents,including mice, rats, and the like. Preferably, the subject is a mammalor a mammalian cell. More preferably, the subject is a human or a humancell. Human subjects include, but are not limited to, fetal, neonatal,infant, juvenile, and adult subjects. Further, a “subject” can include apatient afflicted with or suspected of being afflicted with a conditionor disease. Thus, the terms “subject” and “patient” are usedinterchangeably herein. A subject also can refer to cells or collectionsof cells in laboratory or bioprocessing culture in tests for viability,differentiation, marker production, expression, and the like.

The presently disclosed methods can be used to diagnose, for theprognosis, or the monitoring of a disease state or condition. As usedherein, the term “diagnosis” refers to a predictive process in which thepresence, absence, severity or course of treatment of a disease,disorder or other medical condition is assessed. For purposes herein,diagnosis also includes predictive processes for determining the outcomeresulting from a treatment. Likewise, the term “diagnosing,” refers tothe determination of whether a sample specimen exhibits one or morecharacteristics of a condition or disease. The term “diagnosing”includes establishing the presence or absence of, for example, a targetantigen or reagent bound targets, or establishing, or otherwisedetermining one or more characteristics of a condition or disease,including type, grade, stage, or similar conditions. As used herein, theterm “diagnosing” can include distinguishing one form of a disease fromanother. The term “diagnosing” encompasses the initial diagnosis ordetection, prognosis, and monitoring of a condition or disease.

The term “prognosis,” and derivations thereof, refers to thedetermination or prediction of the course of a disease or condition. Thecourse of a disease or condition can be determined, for example, basedon life expectancy or quality of life. “Prognosis” includes thedetermination of the time course of a disease or condition, with orwithout a treatment or treatments. In the instance where treatment(s)are contemplated, the prognosis includes determining the efficacy of atreatment for a disease or condition.

As used herein, the term “risk” refers to a predictive process in whichthe probability of a particular outcome is assessed.

The term “monitoring,” such as in “monitoring the course of a disease orcondition,” refers to the ongoing diagnosis of samples obtained from asubject having or suspected of having a disease or condition.

The term “marker” refers to a molecule, such as a protein, including anantigen, that when detected in a sample is characteristic of orindicates the presence of a disease or condition.

The presently disclosed subject matter also provides methods formonitoring disease states in a subject, including chronic diseases, suchas, but not limited to, heart disease, coronary artery disease,diabetes, metabolic disorders, inflammatory diseases, such as rheumatoidarthritis, and cancer. The metabolic disorders can include, but are notlimited to, hyperlipidemia, hypolipidemia, hyperthyroidism, andhypothyroidism.

Further, the presently disclosed methods can be used to monitor specificmarkers of a chronic disease. By monitoring the concentrations ofmolecular artifacts, metabolites, and deleterious and/or beneficialmolecules of a disease state, the subject's progression, regression orstability can be assessed, and treatments can, in turn be adjusted orrevised accordingly. For example, markers for heart disease that couldbe monitored in vivo using the presently disclosed biosensors include,but are not limited to, total fatty acids, lactate, glucose, free fattyacids and various cardiotonic agents, such as, but not limited tocardioglycosides and sympathomimetics. Markers of diabetes include, butare not limited to, glucose, lactate and fatty acids. Likewise, markersfor coronary artery disease include, but are not limited to, C-reactivepeptide and free fatty acids. Generally, markers of various metabolicdisorders include, but are not limited to, specific fatty acids.

The presently disclosed SERS-active nanoparticles also are suitable foruse in devices for monitoring drug treatment. Indeed, the SERS-activenanoparticle can be designed to specifically bind a drug, drug candidateor a drug metabolite. In this manner, the plasma concentration of thedrug could be monitored and dosages could be adjusted or maintainedbased on the concentration measurements provided by the SERS method.Accordingly, a pharmaceutical regimen could be individualized for aparticular subject, including the use of a SERS-active nanoparticle thatcan specifically and reversibly bind the drug or drug metabolite todetermine plasma concentrations of the drug. The concentrations providedby the SERS method can then be used to determine the bioavailability ofthe drug in the subject. The dose of the drug administered to thesubject can then be altered to increase or decrease the bioavailabilityof the drug to the subject to provide maximum therapeutic benefits andavoiding toxicity.

The presently disclosed SERS-active nanoparticles also can be used tosimultaneously monitor a variety of metabolites, the measurements ofwhich could be used to profile the subject's metabolic or physicalstate. For example, during extended periods of strenuous exercise,glucose is broken down in anaerobic processes to lactic acid. Thepresently disclosed SERS-active nanoparticles can be used to determinelactate thresholds of athletes, to maximize the benefits of training anddecrease recovery time. Similarly, the SERS-active nanoparticles can beused to determine lactate thresholds in soldiers to prevent fatigue andexhaustion and to decrease recovery time. To that end, the presentlydisclosed SERS-active nanoparticles can be used to monitor glucoselevels, lactic acids levels and other metabolites during exercise orphysical stress.

The presently disclosed SERS-active nanoparticles also can be used tomonitor a condition or disease state in a patient in an acute carefacility, such as an emergency room or a post-operative recovery room ora hospital. For example, in embodiments providing a method formonitoring glucose levels in a subject, studies have shown thatmortality can be decreased by as much as 30% in post-operative patientswhen glucose levels are monitored and kept normal. Thus, the presentlydisclosed SERS-based diagnostic assays can be used in situations wheremonitoring glucose or other metabolites is essential to recovery or theoverall health of the subject.

The amount of one or more analytes present in a sample under test can berepresented as a concentration. As used herein, the term “concentration”has its ordinary meaning in the art. The concentration can be expressedas a qualitative value, for example, as a negative- or positive-typeresult, e.g., a “YES” or “NO” response, indicating the presence orabsence of a target analyte, or as a quantitative value. Further, theconcentration of a given analyte can be reported as a relative quantityor an absolute quantity, e.g., as a “quantitative value.” The presentlydisclosed assays, in some embodiments, are capable of detecting ananalyte of interest at a concentration range of about 5 fg/mL to about500 ng/mL; in some embodiments, at a concentration range of about 10fg/mL to about 100 ng/mL; in some embodiments, at a concentration rangeof about 50 fg/mL to about 50 ng/mL.

The quantity (concentration) of an analyte can be equal to zero,indicating the absence of the particular analyte sought or that theconcentration of the particular analyte is below the detection limits ofthe assay. The quantity measured can be the SERS signal without anyadditional measurements or manipulations. Alternatively, the quantitymeasured can be expressed as a difference, percentage or ratio of themeasured value of the particular analyte to a measured value of anothercompound including, but not limited to, a standard or another analyte.The difference can be negative, indicating a decrease in the amount ofmeasured analyte(s). The quantities also can be expressed as adifference or ratio of the analyte(s) to itself, measured at a differentpoint in time. The quantities of analytes can be determined directlyfrom a generated signal, or the generated signal can be used in analgorithm, with the algorithm designed to correlate the value of thegenerated signals to the quantity of analyte(s) in the sample.

The presently disclosed SERS-active nanoparticles are amenable for usewith devices capable of continuously measuring the concentrations of oneor more analytes. As used herein, the term “continuously,” inconjunction with the measuring of an analyte, is used to mean the deviceeither generates or is capable of generating a detectable signal at anytime during the life span of the device. The detectable signal can beconstant, in that the device is always generating a signal, even if asignal is not detected. Alternatively, the device can be usedepisodically, such that a detectable signal can be generated, anddetected, at any desired time.

B. Cellular Imaging

The small size of the presently disclosed SERS-active nanoparticlesallow the nanoparticles to be incorporated into cells. For example, theuse of SERS to study the complexation of a chemotherapeutic agent withDNA has been demonstrated. See Nabiev, I. R., et al., “Selectiveanalysis of antitumor drug interactions with living cancer cells asprobed by surface-enhanced Raman spectroscopy, Eur. Biophys. J., 19,311-316 (1991); Morjani, H., et al., “Molecular and cellularinteractions between intoplicine, DNA, and topoisomerase II studied bysurface-enhanced Raman scattering spectroscopy,” Cancer Res., 53,4784-4790 (1993). SERS also has been used to investigate the mechanismof chemotherapeutic resistance to certain cancers. See Breuzard, G., etal., “Surface-enhanced Raman scattering reveals adsorption ofmitoxantrone on plasma membrane of living cells,” Biochem. Biophys. Res.Comm., 320, 615-621 (2004). Further, SERS has been used to characterizethe distribution of particular chemicals within cells and to distinguishbetween the cytoplasm and the nucleus of the cell. See Kneipp, K., etal., “Surface-enhanced Raman spectroscopy in single living cells usinggold nanoparticles,” Appl. Spectrosc., 56(2), 150-154 (2002).

Accordingly, in some embodiments, nanoparticles labeled with thepresently disclosed dyes can be used for cellular imaging, for example,to distinguish between abnormal cells, for example, a cell exhibiting ananomaly, such as a cancerous cell, versus normal cells in a biologicalsample. In such embodiments, the intensity of the Raman signal arisingfrom the dye is proportional to the density of cells detected. Further,in some embodiments, the nanoparticles labeled with the presentlydisclosed dyes also can be labeled with another species, such as aspecific binding member of a binding pair, for example, an antibody, tofacilitate binding to a cell of interest. The use of SERS-activenanoparticles for cellular imaging is described in U.S. PatentApplication Publication Nos. 2006/0054506 and 2006/0046313, each ofwhich is incorporated herein by reference in its entirety.

Accordingly, in some embodiments, the presently disclosed subject matterprovides a method for detecting the presence of one or more targetstructures in a sample cell, the method comprising:

(a) contacting one or more sample cells with one or more SERS-activenanoparticles labeled with one or more binding members under conditionssuitable for binding of the one or more binding members to one or moretarget structures in the sample cell, wherein the SERS-activenanoparticle has associated therewith a dye of Formula A-Y capable ofproducing a distinguishable Raman signal:

A-Y

wherein:

A is selected from the group consisting of:

wherein X₁ is CR₄ or N;

Y is selected from the group consisting of:

wherein:

r, s, and t are each independently an integer from 1 to 8;

each X₂ and X₃ is independently selected from the group consisting of C,S, and N, under the proviso that (i) when X₂ is C or S, R₅ is Z, or whenX₃ is C or S, R₆ is Z, as Z is defined herein below; (ii) if both X₂ andX₃ are N at the same time, at least one of R₅ and R₆ is absent; and(iii) when X₂ is N, R₅ when present is Z′, or when X₃ is N, R₆ whenpresent is Z′, wherein Z′ is selected from the group consisting of:

—(CH₂)_(n)—X₄; —NR₈—(CH₂)_(p)—X₅; —(CH₂)_(q)X₆C(═O)—R₉,

wherein:

n, p, q, u, and v are each independently an integer from 1 to 8;

X₄ and X₅ are each independently selected from the group consisting ofhydroxyl, amino, and thiol;

X₆ is O or NR₁₁;

wherein:

each R₁, R₂, R₃, R₄, R₅, R₆, R₈, R₁₀, R₁₁, and Z is independentlyselected from the group consisting of H, alkyl, substituted alkyl,heteroalkyl, substituted heteroalkyl, cycloalkyl, substitutedcycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl,substituted aryl, aralkyl, hydroxyl, alkoxyl, hydroxyalkyl,hydroxycycloalkyl, alkoxycycloalkyl, aminoalkyl, acyloxyl,alkylaminoalkyl, and alkoxycarbonyl;

R₇ is Z′;

R₉ is —(CH₂)_(m)—X₇ or —(CH₂)_(m)—B, wherein

m is an integer from 1 to 8;

X₇ is halogen; and

B is a binding member having a binding affinity for a ligand or analyteto be detected; and

(b) detecting one or more distinguishable SERS signals from the samplecell to indicate the presence of the one or more target structures inthe sample cell.

In some embodiments, the method further comprises contacting the one ormore SERS-active nanoparticles with one or more reference cells. In someembodiments, the method further comprises analyzing the one of moredistinguishable SERS signals detected from the sample cell to constructa profile of the one or more target structures in the sample cell. Insome embodiments, the method further comprises analyzing the one or moredistinguishable SERS signals detected from the reference cell toconstruct a profile of the one or more target structures in thereference cell. In some embodiments, the method further comprisescomparing the profile of the one or more target structures in the samplecell to the profile of the one or more target structures in thereference cell. In some embodiments, a difference in the profile of thesample cell as compared to the profile of the reference cell isindicative of an anomaly of the sample cell.

In some embodiments, the presently disclosed SERS-active nanoparticlescan be used for staining microstructures within a cell. In suchembodiments, the SERS-active nanoparticles can be labeled with at leastone ligand that specifically binds to a known target microstructure orreceptor. In some embodiments, a set of SERS-active nanoparticle probescan be used, wherein each member of the set comprises a combination of aligand that specifically binds to a known target or receptor and one ormore SERS-active dyes that can produce a distinguishable SERS signalupon binding with the target.

Under suitable conditions, the labeled SERS-active nanoparticles canspecifically bind to receptors and other microstructures within thecell. The “stained” cells can then be imaged, for example, by using ascanning Raman microscope to determine the presence and location ofspecific receptors and microstructures in the cells. Further, the SERSsignals from individual Raman-active dyes associated with a particularligand can be used to distinguish between specific receptors andmicrostructures in the cell and to create a profile of the receptors andmicrostructures in the cell. The profile of a target cell assayedaccording to the presently disclosed method can be compared with aprofile similarly obtained from a normal cell of the same type todetermine the presence of an anomaly in the target cell. The target cellcan be either living or dead.

As used herein, the term “microstructure” includes, but is not limitedto, extracellular matrix molecules, such as fibronectin and laminin;intracellular structures, such as actin filaments and microtubes; cellnucleus structures, such as histone; and the like. Suitable ligands forbinding to such microstructures can be selected from the ligandsdisclosed herein, and include, but are not limited to, antibodies, suchas anti-fibronectin antibodies and anti-actin antibodies, and othernaturally-occurring ligands, such as anti-histone protein.

Images of cells containing Raman spectral information can be obtained bya variety of methods known in the art. For example, a microscope can becoupled to a charge-coupled device (CCD) camera such that completeimages of the sample can be obtained. Typically, in such embodiments, awavenumber (or wavelength) filtering device, such as a monochromator orliquid crystal tunable filter, can be inserted between the sample andthe CCD camera. The filtering device allows only a narrow bandwidth ofscattered radiation to reach the CCD camera at any one time. Multipleimages can be collected by the CCD camera, wherein each image covers aparticular spectral range of the scattered radiation. The spectra fromeach point in the image can be assembled in software. Alternatively,light from a single point of an image can be dispersed through amonochromator and the complete spectrum of that point can be acquired onan array detector. The sample can be scanned such that each point in theimage is acquired separately. The Raman image is then assembled insoftware. In another approach, a line scan instrument can be constructedthat excites the sample with a line of radiation. The line is imagedspatially along one axis of a CCD camera while simultaneously beingspectrally dispersed along the orthogonal axis. Each readout of thecamera acquires the complete spectrum of each spatial pixel in the line.To complete the image the line is scanned across the sample. An exampleof a Raman instrument suitable for imaging is described in Talley, etal., “Nanoparticle Based Surface-Enhanced Raman Spectroscopy,” NATOAdvanced Study Institute: Biophotonics, Ottawa, Canada (Jan. 6, 2005).

In some embodiments, the presently disclosed SERS-active nanoparticlescan be incorporated into a cell or tissue by a passive uptake mechanism.Another mechanism for incorporating nanoparticles into cells is throughthe use of small peptide, which can bind to endocytotic receptors on thecell surface and draw the nanoparticles into the cell throughendocytosis. See Tkachenko, A. G., et al., “Cellular trajectories ofpeptide-modified gold particle complexes: comparison of nuclearlocalization signals and peptide transduction domains,” BioconjugateChem., 15, 482-490 (2004). Further, the SERS-active nanoparticles can beintroduced into cells via microinjection, transfection, electroporation,and endocytosis-mediated approaches, including the use of amphipathicpeptides, such as PEP-1, the use of cationic lipid-based reagents, suchas LIPOFECTAMINE™ (Invitrogen Corp., Carlsbad, Calif., United States ofAmerica), and the use of micelles and transfection reagents such astransferrin, mannose, galactose, and Arg-Gly-Asp (RGD), and otherreagents such as the dendrimer-based reagent SUPERFECT™ (Qiagen, Inc.,Valencia, Calif., United States of America). Intracellularly, indirectmethods can be used to show that the particles are bound to the desiredtargets. One method suitable for demonstrating the specificity of theprobes is immunofluorescence, which can be used to verify the locationof the SERS-active nanoparticles. A number of commercially availablefluorescent probes are useful for labeling cellular structures (such asthe mitochondria, Golgi apparatus and endoplasmic reticulum) in livingcells. By conjugating an antibody that targets the same structure, thefraction of nanoparticles that actively label their target can bedetermined. Likewise, what percentage of nanoparticles that arenon-specifically bound also can be determined. Another approach toverifying the location of the SERS-active nanoparticles is to usefluorescent protein fusions, such as GFP and its analogs.

In some embodiments, imaging agents comprising the presently disclosedSERS-active nanoparticles are provided for use in medical diagnosis. Thepresently disclosed imaging agents are useful in imaging a patientgenerally, and/or specifically diagnosing the presence of diseasedtissue in a patient. As described hereinabove, by selecting the size,shape, and composition of the nanoparticle core; the identity of thedye; and the composition and thickness of encapsulant, if desired, theoptimum excitation and emission frequencies of the SERS-activenanoparticles can be tuned to occur between about 630 nm and about 1000nm, i.e., the minimum region for absorption and scattering by tissues.

An imaging process can be carried out by administering an imaging agentcomprising one or more presently disclosed SERS-active nanoparticles toa cell, a tissue sample, or to a subject, such as a patient, and thenscanning the cell, tissue sample, or subject using any system known inthe art that can perform spectral imaging, including, but not limited tospot scanning confocal microscopes, line scanning systems, and OpticalCoherence tomographic systems. The presence of the presently disclosedSERS-active nanoparticle in a cell, tissue sample, or subject also canbe observed by any imaging systems that detects over a single wavelengthband, as well as any fluorescence imaging system that includes anexcitation light source and filtered image detection. Other imagingsystems suitable for use with the presently disclosed SERS-activenanoparticles are described in Tuchin, V. V., Handbook of opticalbiomedical diagnostics, Bellingham, Wash., USA: SPIE Press, 2002, whichis included herein by reference in its entirety. Other imaging methods,including time domain methods, such as dynamic light scatteringspectroscopy and tomography, time-of-flight imaging, quasi-elastic lightscattering spectroscopy, photon-correlation spectroscopy, Dopplerspectroscopy, and diffusion wave spectroscopy are suitable for use withthe presently disclosed subject matter. All these techniques allowdifferentiation between photons and where they have been based on theirtime signatures. Because SERS-active nanoparticles can have differenttime signatures than fluorescent substances and the like, they can bediscriminated against tissues and other labels with these methods.Useful instrument parameters also include a modulated light source andtime sensitive detector. The modulation can be pulsed or continuous.

The scanning of the cell, tissue sample, or subject provides spectra orimages of an internal region of the cell, tissue sample, or subject andcan be used to detect or diagnose the presence of a condition or adisease state. By region of a cell, tissue sample, or subject, it ismeant the whole cell, tissue sample, or subject, or a particular area orportion of the cell, tissue sample, or subject. When the subject is apatient, the presently disclosed imaging agents can be used to provideimages of internal organs of the patient, including vasculature, heart,liver, and spleen, and in imaging the gastrointestinal region or otherbody cavities, or in other ways as will be readily apparent to thoseskilled in the art, such as in tissue characterization, blood poolimaging, and the like.

The presently disclosed subject matter also provides, in someembodiments, a method of diagnosing abnormal pathology in vivo, themethod including introducing a plurality of SERS-active nanoparticlestargeted to a molecule involved in the abnormal pathology into a bodilyfluid contacting the abnormal pathology, wherein the SERS-activenanoparticles can become associated with the molecule involved in theabnormal pathology, and imaging the associated SERS-active nanoparticlesin vivo. The presently disclosed method is generally applicable to anyorgan accessible by the SERS-active nanoparticle probes, including thegastrointestinal tract, heart, lung, liver cervix, breast, and the like.

In some embodiments, the presently disclosed SERS-active nanoparticlescan be introduced into a subject via an endoscope, as in the case of acolonoscopy, or a needle, or used with a disposable tip or sleeve, orvia endocytosis, transfection, microinjection, and the like. In otherembodiments, the SERS-active nanoparticle probes can be introduced bydirectly introducing the imaging probe itself. In some embodiments,individual optical fibers, or bundles of optical fibers, can beintroduced into live organisms for imaging. Such methods have beendemonstrated for imaging of nerves, brain, microvessels, cells, as wellas for characterizing biodistribution. Gel-coated optical fibers arewell known in the sensor literature. The presently disclosed SERS-activenanoparticles can be non-covalently bound to the gel, wherein thenanoparticles can diffuse into the tissue upon introduction into thetissue. A variety of other methods to immobilize SERS-activenanoparticles on the outer surface of fibers such that they can diffuseinto liquid phases to which they are contacted also are suitable for usewith the presently disclosed subject matter.

In some embodiments, the presently disclosed subject matter provides amethod for labeling an animal with a SERS-active nanoparticle, themethod comprising introducing a SERS-active nanoparticle into theanimal. The presently disclosed SERS-active nanoparticles can beintroduced into an animal by any suitable method, including, but notlimited to, any subcutaneous implantation method or intravenously. TheSERS-active nanoparticle can be detected using appropriateinstrumentation. In some embodiments, the presently disclosed subjectmatter provides an identification system for animals, includinglivestock and domesticated pets, wherein the SERS-active nanoparticle isimplanted under the skin (or hide) of the animal to enableidentification.

III. Chemical Definitions

While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the presently disclosed subject matter. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this presently described subject matter belongs.

Throughout the specification and claims, a given chemical formula orname shall encompass all optical and stereoisomers, as well as racemicmixtures where such isomers and mixtures exist.

When the term “independently selected” is used, the substituents beingreferred to (e.g., R groups, such as groups R₁, R₂, and the like, orgroups X₁ and X₂), can be identical or different. For example, both R₁and R₂ can be substituted alkyls, or R₁ can be hydrogen and R₂ can be asubstituted alkyl, and the like.

A named “R” or “X” group will generally have the structure that isrecognized in the art as corresponding to a group having that name,unless specified otherwise herein. For the purposes of illustration,certain representative “R” and “X” groups as set forth above are definedbelow. These definitions are intended to supplement and illustrate, notpreclude, the definitions that would be apparent to one of ordinaryskill in the art upon review of the present disclosure.

As used herein the term “alkyl” refers to C₁₋₂₀ inclusive, linear (i.e.,“straight-chain”), branched, or cyclic, saturated or at least partiallyand in some cases fully unsaturated (i.e., alkenyl andalkynyl)hydrocarbon chains, including for example, methyl, ethyl,propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, octyl,ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl,propynyl, butynyl, pentynyl, hexynyl, heptynyl, and alkenyl groups.“Branched” refers to an alkyl group in which a lower alkyl group, suchas methyl, ethyl or propyl, is attached to a linear alkyl chain. “Loweralkyl” refers to an alkyl group having 1 to about 8 carbon atoms (i.e.,a C₁₋₈ alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. “Higheralkyl” refers to an alkyl group having about 10 to about 20 carbonatoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms.In certain embodiments, “alkyl” refers, in particular, to C₁₋₈straight-chain alkyls. In other embodiments, “alkyl” refers, inparticular, to C₁₋₈ branched-chain alkyls.

Alkyl groups can optionally be substituted (a “substituted alkyl”) withone or more alkyl group substituents, which can be the same ordifferent. The term “alkyl group substituent” includes but is notlimited to alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl,aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio,carboxyl, alkoxycarbonyl, oxo, and cycloalkyl. There can be optionallyinserted along the alkyl chain one or more oxygen, sulfur or substitutedor unsubstituted nitrogen atoms, wherein the nitrogen substituent ishydrogen, lower alkyl (also referred to herein as “alkylaminoalkyl”), oraryl.

Thus, as used herein, the term “substituted alkyl” includes alkylgroups, as defined herein, in which one or more atoms or functionalgroups of the alkyl group are replaced with another atom or functionalgroup, including for example, alkyl, substituted alkyl, halogen, aryl,substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino,dialkylamino, sulfate, and mercapto.

“Cyclic” and “cycloalkyl” refer to a non-aromatic mono- or multicyclicring system of about 3 to about 10 carbon atoms, e.g., 3, 4, 5, 6, 7, 8,9, or 10 carbon atoms. The cycloalkyl group can be optionally partiallyunsaturated. The cycloalkyl group also can be optionally substitutedwith an alkyl group substituent as defined herein, oxo, and/or alkylene.There can be optionally inserted along the cyclic alkyl chain one ormore oxygen, sulfur or substituted or unsubstituted nitrogen atoms,wherein the nitrogen substituent is hydrogen, alkyl, substituted alkyl,aryl, or substituted aryl, thus providing a heterocyclic group.Representative monocyclic cycloalkyl rings include cyclopentyl,cyclohexyl, and cycloheptyl. Multicyclic cycloalkyl rings includeadamantyl, octahydronaphthyl, decalin, camphor, camphane, andnoradamantyl.

The term “cycloalkylalkyl,” as used herein, refers to a cycloalkyl groupas defined hereinabove, which is attached to the parent molecular moietythrough an alkyl group, also as defined above. Examples ofcycloalkylalkyl groups include cyclopropylmethyl and cyclopentylethyl.

The terms “cycloheteroalkyl” or “heterocycloalkyl” refer to anon-aromatic ring system, such as a 3- to 7-member substituted orunsubstituted cycloalkyl ring system, including one or more heteroatoms,which can be the same or different, and are selected from the groupconsisting of N, O, and S, and optionally can include one or more doublebonds. The cycloheteroalkyl ring can be optionally fused to or otherwiseattached to other cycloheteroalkyl rings and/or non-aromatic hydrocarbonrings. Representative cycloheteroalkyl ring systems include, but are notlimited to pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl,pyrazolidinyl, pyrazolinyl, piperidyl, piperazinyl, indolinyl,quinuclidinyl, morpholinyl, thiomorpholinyl, thiadiazinanyl,tetrahydrofuranyl, and the like.

The term “alkenyl” as used herein refers to a straight or branchedhydrocarbon of a designed number of carbon atoms containing at least onecarbon-carbon double bond. Examples of “alkenyl” include vinyl, allyl,2-methyl-3-heptene, and the like.

The term “cycloalkenyl” as used herein refers to a cyclic hydrocarboncontaining at least one carbon-carbon double bond. Examples ofcycloalkenyl groups include cyclopropenyl, cyclobutenyl, cyclopentenyl,cyclopentadiene, cyclohexenyl, 1,3-cyclohexadiene, cycloheptenyl,cycloheptatrienyl, and cyclooctenyl.

The term “alkynyl” as used herein refers to a straight or branchedhydrocarbon of a designed number of carbon atoms containing at least onecarbon-carbon triple bond. Examples of “alkynyl” include propargyl,propyne, and 3-hexyne.

“Alkylene” refers to a straight or branched bivalent aliphatichydrocarbon group having from 1 to about 20 carbon atoms, e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbonatoms. The alkylene group can be straight, branched or cyclic. Thealkylene group also can be optionally unsaturated and/or substitutedwith one or more “alkyl group substituents.” There can be optionallyinserted along the alkylene group one or more oxygen, sulfur orsubstituted or unsubstituted nitrogen atoms (also referred to herein as“alkylaminoalkyl”), wherein the nitrogen substituent is alkyl aspreviously described. Exemplary alkylene groups include methylene(—CH₂—); ethylene (—CH₂—CH₂—); propylene (—(CH₂)₃—); cyclohexylene(—C₆H₁₀—); —CH═CH—CH═CH—; —CH═CH—CH₂—; —(CH₂)_(q)—N(R)—(CH₂)_(r)—,wherein each of q and r is independently an integer from 0 to about 20,e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20, and R is hydrogen or lower alkyl; methylenedioxyl(—O—CH₂—O—); and ethylenedioxyl (—O—(CH₂)₂—O—). An alkylene group canhave about 2 to about 3 carbon atoms and can further have 6-20 carbons.

The term “aryl” is used herein to refer to an aromatic substituent thatcan be a single aromatic ring, or multiple aromatic rings that are fusedtogether, linked covalently, or linked to a common group, such as, butnot limited to, a methylene or ethylene moiety. The common linking groupalso can be a carbonyl, as in benzophenone, or oxygen, as indiphenylether, or nitrogen, as in diphenylamine. The term “aryl”specifically encompasses heterocyclic aromatic compounds. The aromaticring(s) can comprise phenyl, naphthyl, biphenyl, diphenylether,diphenylamine and benzophenone, among others. In particular embodiments,the term “aryl” means a cyclic aromatic comprising about 5 to about 10carbon atoms, e.g., 5, 6, 7, 8, 9, or 10 carbon atoms, and including 5-and 6-membered hydrocarbon and heterocyclic aromatic rings.

The aryl group can be optionally substituted (a “substituted aryl”) withone or more aryl group substituents, which can be the same or different,wherein “aryl group substituent” includes alkyl, substituted alkyl,aryl, substituted aryl, aralkyl, hydroxyl, alkoxyl, aryloxyl,aralkyloxyl, carboxyl, acyl, halo, nitro, alkoxycarbonyl,aryloxycarbonyl, aralkoxycarbonyl, acyloxyl, acylamino, aroylamino,carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, arylthio, alkylthio,alkylene, and —NR′R″, wherein R′ and R″ can each be independentlyhydrogen, alkyl, substituted alkyl, aryl, substituted aryl, and aralkyl.

Thus, as used herein, the term “substituted aryl” includes aryl groups,as defined herein, in which one or more atoms or functional groups ofthe aryl group are replaced with another atom or functional group,including for example, alkyl, substituted alkyl, halogen, aryl,substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino,dialkylamino, sulfate, and mercapto.

Specific examples of aryl groups include, but are not limited to,cyclopentadienyl, phenyl, furan, thiophene, pyrrole, pyran, pyridine,imidazole, benzimidazole, isothiazole, isoxazole, pyrazole, pyrazine,triazine, pyrimidine, quinoline, isoquinoline, indole, carbazole, andthe like.

The term “heteroaryl” refers to an aromatic ring system, such as, butnot limited to a 5- or 6-member ring system, including one or moreheteroatoms, which can be the same or different, and are selected fromthe group consisting of N, O, and S. The heteroaryl ring can be fused orotherwise attached to one or more heteroaryl rings, aromatic ornon-aromatic hydrocarbon rings, or heterocycloalkyl rings.Representative heteroaryl ring systems include, but are not limited to,pyridyl, pyrimidyl, pyrrolyl, pyrazolyl, azolyl, oxazolyl, isoxazolyl,oxadiazolyl, thiazolyl, isothiazolyl, imidazolyl, furanyl, thienyl,quinolinyl, isoquinolinyl, indolinyl, indolyl, benzothienyl,benzothiazolyl, enzofuranyl, benzimidazolyl, benzisoxazolyl,benzopyrazolyl, triazolyl, tetrazolyl, and the like.

A structure represented generally by the formula, wherein the ringstructure can be aromatic or non-aromatic:

as used herein refers to a ring structure, for example, but not limitedto a 3-carbon, a 4-carbon, a 5-carbon, a 6-carbon, and the like,aliphatic and/or aromatic cyclic compound, including a saturated ringstructure, a partially saturated ring structure, and an unsaturated ringstructure as defined herein, comprising a substituent R group, whereinthe R group can be present or absent, and when present, one or more Rgroups can each be substituted on one or more available carbon atoms ofthe ring structure. The presence or absence of the R group and number ofR groups is determined by the value of the integer n. Each R group, ifmore than one, is substituted on an available carbon of the ringstructure rather than on another R group. For example, the structureabove where n is 0 to 2 would comprise compound groups including, butnot limited to:

and the like.

A dashed line representing a bond in a cyclic ring structure indicatesthat the bond can be either present or absent in the ring. That is, adashed line representing a bond in a cyclic ring structure indicatesthat the ring structure is selected from the group consisting of asaturated ring structure, a partially saturated ring structure, and anunsaturated ring structure.

When a named atom of an aromatic ring or a heterocyclic aromatic ring isdefined as being “absent,” the named atom is replaced by a direct bond.

As used herein, the term “acyl” refers to an organic acid group whereinthe —OH of the carboxyl group has been replaced with another substituent(i.e., as represented by RCO—, wherein R is an alkyl or an aryl group asdefined herein). As such, the term “acyl” specifically includes arylacylgroups, such as an acetylfuran and a phenacyl group. Specific examplesof acyl groups include acetyl and benzoyl.

“Alkoxyl” refers to an alkyl-O— group wherein alkyl is as previouslydescribed. The term “alkoxyl” as used herein can refer to C₁₋₂₀inclusive, linear, branched, or cyclic, saturated or unsaturatedoxo-hydrocarbon chains, including, for example, methoxyl, ethoxyl,propoxyl, isopropoxyl, butoxyl, t-butoxyl, and pentoxyl.

The term “alkoxyalkyl” as used herein refers to an alkyl-O-alkyl ether,for example, a methoxyethyl or an ethoxymethyl group.

“Aryloxyl” refers to an aryl-O— group wherein the aryl group is aspreviously described, including a substituted aryl. The term “aryloxyl”as used herein can refer to phenyloxyl or hexyloxyl, and alkyl,substituted alkyl, halo, or alkoxyl substituted phenyloxyl or hexyloxyl.

The term “alkyl-thio-alkyl” as used herein refers to an alkyl-S-alkylthioether, for example, a methylthiomethyl or a methylthioethyl group.

“Aralkyl” refers to an aryl-alkyl-group wherein aryl and alkyl are aspreviously described, and included substituted aryl and substitutedalkyl. Exemplary aralkyl groups include benzyl, phenylethyl, andnaphthylmethyl.

“Aralkyloxyl” refers to an aralkyl-O— group wherein the aralkyl group isas previously described. An exemplary aralkyloxyl group is benzyloxyl.“Alkoxycarbonyl” refers to an alkyl-O—CO— group. Exemplaryalkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl,butyloxycarbonyl, and t-butyloxycarbonyl. “Aryloxycarbonyl” refers to anaryl-O—CO-group. Exemplary aryloxycarbonyl groups include phenoxy- andnaphthoxy-carbonyl. “Aralkoxycarbonyl” refers to an aralkyl-O—CO— group.An exemplary aralkoxycarbonyl group is benzyloxycarbonyl.

“Carbamoyl” refers to an H₂N—CO— group. “Alkylcarbamoyl” refers to aR′RN—CO— group wherein one of R and R′ is hydrogen and the other of Rand R′ is alkyl and/or substituted alkyl as previously described.“Dialkylcarbamoyl” refers to a R′RN—CO— group wherein each of R and R′is independently alkyl and/or substituted alkyl as previously described.

“Acyloxyl” refers to an acyl-O— group wherein acyl is as previouslydescribed.

The term “amino” refers to the —NH₂ group and also refers to a nitrogencontaining group as is known in the art derived from ammonia by thereplacement of one or more hydrogen radicals by organic radicals. Forexample, the terms “acylamino” and “alkylamino” refer to specificN-substituted organic radicals with acyl and alkyl substituent groupsrespectively.

The term “alkylamino” refers to an —NHR group wherein R is an alkylgroup and/or a substituted alkyl group as previously described.Exemplary alkylamino groups include methylamino, ethylamino, and thelike.

“Dialkylamino” refers to an —NRR′ group wherein each of R and R′ isindependently an alkyl group and/or a substituted alkyl group aspreviously described. Exemplary dialkylamino groups includeethylmethylamino, dimethylamino, and diethylamino.

“Acylamino” refers to an acyl-NH— group wherein acyl is as previouslydescribed. “Aroylamino” refers to an aroyl-NH— group wherein aroyl is aspreviously described.

The term “carbonyl” refers to the —(C═O)— group.

The term “carboxyl” refers to the —COOH group.

The terms “halo”, “halide”, or “halogen” as used herein refer to fluoro,chloro, bromo, and iodo groups.

The term “hydroxyl” refers to the —OH group.

The term “hydroxyalkyl” refers to an alkyl group substituted with an —OHgroup.

The term “mercapto” refers to the —SH group.

The term “oxo” refers to a compound described previously herein whereina carbon atom is replaced by an oxygen atom.

The term “nitro” refers to the —NO₂ group.

The term “thio” refers to a compound described previously herein whereina carbon or oxygen atom is replaced by a sulfur atom. For example, a“thiol” group refers to the group —SH.

The term “sulfate” refers to the —SO₄ group.

EXAMPLES

The following Examples have been included to provide guidance to one ofordinary skill in the art for practicing representative embodiments ofthe presently disclosed subject matter. In light of the presentdisclosure and the general level of skill in the art, those of skill canappreciate that the following Examples are intended to be exemplary onlyand that numerous changes, modifications, and alterations can beemployed without departing from the scope of the presently disclosedsubject matter. The following Examples are offered by way ofillustration and not by way of limitation.

Example 1 Preparation of Nanoparticles Having Near-Infrared DyesAttached Thereto

Spherical gold nanoparticles with a diameter of 60 nm were purchasedfrom Ted Pella, Inc. (Redding, Calif., United States of America). Thesize of the nanoparticle as reported by the manufacturer was confirmedby transmission electron microscopy and light scattering. To attachRaman active species, e.g., non-fluorescent molecules and the presentlydisclosed NIR dyes, to the gold nanoparticles, the chemicals were mixedwith gold nanoparticles such that the concentration of the Ramanreporter was 10 μM in the final mixture. The gold nanoparticles wereconcentrated five times from their bulk solution to provide a finaloptical density equal to five (5) at 520 nm. The reaction was allowed toproceed overnight on a shaker.

Example 2 SERS Spectra of Near-Infrared Dyes

The instrument used for testing the SERS intensity of the presentlydisclosed SERS-active dyes was a Centice (Morrisville, N.C., UnitedState of America) spectrometer with 660 nm laser excitation. The resultsof the experiments for a commonly-used non-fluorescent Raman activemolecule, e.g., trans-1,2-bis(4-pyridyl)ethylene (BPE) and a presentlydisclosed NIR dye, e.g., Coumarin picolinium dye (CoPic) appear inFIG. 1. BPE exhibits two major peaks: one peak at approximately 1200cm⁻¹ and a doublet between 1600 and 1650 cm⁻¹. Similarly, CoPic exhibitstwo major peaks at approximately 1150 and 1550 cm⁻¹. While the maximumintensity of the most prominent peak in the BPE spectrum is about47,000, the maximum for CoPic is about 160,000. In this example, thepeak maximum for CoPic is approximately three times as intense as thatfrom BPE.

SERS data obtained for a number of commercially availablenon-fluorescent Raman molecules and dyes are presented in FIG. 2 andTable 1. In each case, the intensity of the most prominent Raman peakabove the background is reported. All of the commercial dyes, exceptMGITC, do not perform as well as BPE. The intensity of the Raman peaksshown by the presently disclosed NIR dyes determined under similarexperimental conditions is shown in FIG. 3. The structures of the dyesappear in FIG. 4. Some dyes did not show any Raman peaks, whereas otherdyes exhibited intense peaks. FIG. 3 also compares the Raman intensityobtained for some commercially available Raman reporter molecules andthe presently disclosed NIR dyes. In some embodiments, the presentlydisclosed dyes are two to five times more intense that reportermolecules known in the art at 660 nm laser excitation.

TABLE 1 Raman Intensity Observed at Most Intense Peak for RepresentativeCommercially-Available Raman Molecules and Dyes. Intensity ArbitraryPrimary Abbrev- Units Peak No. iation Name Source (a.u.) (cm⁻¹) 1 MBA4-mercaptobenzoic Sigma† 100 1590 acid 2 ATP 4-aminothiophenol Sigma†5500 1528 3 BPE Trans-1,2-bis(4- Sigma† 47000 1608 pyridyl)ethylene 4Cy-5 Cyanine-5 Amersham‡ 100 — 5 Rh6G Rhodamine 6G Sigma† 1500 1457 6FIAm Fluorescamine Sigma† 1500 1599 7 FITC Fluorescein Sigma† 2000 1599isothiocyanate 8 RB Rhodamine B Sigma† 4000 1515 9 Rh101 Rhodamine 101Sigma† 4500 — 10 CV Cresyl violet Sigma† 16000 1638 11 TRITC Tetra-Sigma† 27000 1651 methylrhodamine isothiocyanate 12 RBITC Rhodamine BSigma† 33000 1515 isothiocyanate 13 XRITC X-rhodamine- Molecular 360001648 5-(and-6) Probes ®†† isothiocyanate 14 MGITC Malachite greenMolecular 80000 1623 isothiocyanate Probes ®†† †Sigma-Aldrich Co., St.Louis, Missouri; ‡Amersham Biosciences, Piscataway, New Jersey;††Invitrogen Corporation, Carlsbad, California.

TABLE 2 Raman Intensity Observed at Most Intense Peak for RepresentativePresently Disclosed Near- Infrared Raman Dyes. Primary Intensity PeakNo. Abbreviation Name (a.u.) (cm⁻¹) 15 ERB Eno Red B 100 — 18 AZCOIodoacetyl aza- 85000 1650 coumarin 19 CoBzt Coumarin 119000 1560benzothiazole 20 CoPic Coumarin 162000 1573 picolinium 21 BDCYBenzodioxazole 235000 1555 cyanine

Example 3 Reduction of Non-Specific Binding in Magnetic CaptureLiquid-Based SERS Assays

DNA oligonucleotides were attached to the surface of SERS-activenanoparticles (gold particles coated with SERS-active reportermolecules, e.g., various bipyridyl dyes, and encapsulated by athiol-functionalized glass coating) through a polyethylene glycol (PEG)linker molecule. A representative example of one embodiment of thepresently disclosed subject matter is illustrated in FIG. 5, whichdepicts the immobilization of DNA on the surface of SERS-activenanoparticles and magnetic capture particles via a 5-nmheterobifunctional PEG linker molecule.

The amine-terminated oligonucleotides were reacted with theN-hydroxy-succinimide (NHS) ester moiety of a heterobifunctionalpolyethylene glycol molecule comprising an NHS ester separated from athiol-reactive maleimide group by twelve ethylene glycol subunits.Immobilization of PEGylated DNA on SERS-active nanoparticles wasaccomplished by the reaction of maleimide groups on the PEG linker withthiol groups present on the surface of the SERS-active nanoparticle.

DNA oligonucleotides conjugated to a heterobifunctional PEG moleculewere coupled to magnetic particles through traditional1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) coupling bytreating carboxylated magnetic particles with an amine-terminatedmolecule containing an internal disulfide. The disulfide was thencleaved with dithiothreitol, exposing a reactive thiol group thatreacted with the maleimide moiety of the oligonucleotide-PEG conjugate.

To measure the non-specific binding between the oligonucleotide-coatedSERS-active nanoparticle and the oligonucleotide-coated magneticparticle, 10 μL of a 1-mg/mL solution of oligonucleotide-coated magneticparticles were mixed with 100 μL of a 10-pM solution ofoligonucleotide-coated SERS particles and 100 μL of buffer in a smalltube. The tube was inverted for 30 to 120 minutes, followed byconcentration of the magnetic particles with a magnet to form a pellet.The pellet was subsequently interrogated with a laser to determine thelevel of SERS signal, which is proportional to the number of SERS-activereporter molecules associated with the magnetic particles in the pellet.

FIG. 6A shows one spectrum of a blank sample tube and another ofSERS-active nanoparticles and magnetic particles mixed together, whereina DNA oligonucleotide has been directly attached (no PEG linker) to theSERS-active nanoparticle and the magnetic particle via abiotin-streptavidin strategy. Due to the lack of target DNA (analyte) inthe solution, the observed SERS signal is due to non-specificassociation of the SERS-active nanoparticle with the magnetic particles.FIGS. 6B and 6C show results from a similar assay performed witholigonucleotide-coated SERS-active nanoparticles andoligonucleotide-coated magnetic particles, wherein the oligonucleotidesare attached via a PEG linker and prepared as described above. FIGS. 6Aand 6B are plotted on the same scale. FIG. 6C presents the same data asFIG. 6B, but over a narrower signal range. FIG. 6C highlights thesimilarity between the signal from the tube alone and the assay signal,demonstrating that non-specific binding between the two sets ofparticles has been nearly eliminated.

Although the foregoing subject matter has been described in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be understood by those skilled in the art thatcertain changes and modifications can be practiced within the scope ofthe appended claims.

All publications, patent applications, patents, and other references areherein incorporated by reference to the same extent as if eachindividual publication, patent application, patent, and other referencewas specifically and individually indicated to be incorporated byreference. It will be understood that, although a number of patentapplications, patents, and other references are referred to herein, suchreference does not constitute an admission that any of these documentsforms part of the common general knowledge in the art.

1. A nanoparticle comprising a SERS-active reporter molecule of theformula:A-Y wherein: A is:

wherein X₁ is CR₄ or N; Y is selected from the group consisting of:

wherein: r, s, and t are each independently an integer from 1 to 8; eachX₂ and X₃ is independently selected from the group consisting of C, S,and N, under the proviso that (i) when X₂ is C or S, R₅ is Z, or when X₃is C or S, R₆ is Z, as Z is defined herein below; (ii) if both X₂ and X₃are N at the same time, at least one of R₅ and R₆ is absent; and (iii)when X₂ is N, R₅ when present is Z′, or when X₃ is N, R₆ when present isZ′, wherein Z′ is selected from the group consisting of:—(CH₂)_(n)—X₄; —NR₈—(CH₂)_(p)—X₅; —(CH₂)_(q)X₆C(═O)—R₉,

wherein: n, p, q, u, and v are each independently an integer from 1 to8; X₄ and X₅ are each independently selected from the group consistingof hydroxyl, amino, and thiol; X₆ is O or NR₁₁; wherein: each R₁, R₂,R₃, R₄, R₅, R₆, R₈, R₁₀, R₁₁, and Z is independently selected from thegroup consisting of H, alkyl, substituted alkyl, heteroalkyl,substituted heteroalkyl, cycloalkyl, substituted cycloalkyl,cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl,aralkyl, hydroxyl, alkoxyl, hydroxyalkyl, hydroxycycloalkyl,alkoxycycloalkyl, aminoalkyl, acyloxyl, alkylaminoalkyl, andalkoxycarbonyl; R₇ is Z′; R₉ is —(CH₂)_(m)—X₇ or —(CH₂)_(m)—B, wherein mis an integer from 1 to 8; X₇ is halogen; and B is a binding memberhaving a binding affinity for a ligand or analyte to be detected.
 2. TheSERS active nanoparticle of claim 1, wherein the SERS-active reportermolecule of formula A-Y is selected from the group consisting of:

wherein: X′ is selected from the group consisting of hydroxyl, amino,and thiol; and each R₁ and R₂ is independently selected from the groupconsisting of H, alkyl, substituted alkyl, heteroalkyl, substitutedheteroalkyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl,substituted cycloheteroalkyl, aryl, substituted aryl, aralkyl, hydroxyl,alkoxyl, hydroxyalkyl, hydroxycycloalkyl, alkoxycycloalkyl, aminoalkyl,acyloxyl, alkylaminoalkyl, and alkoxycarbonyl.
 3. The SERS activenanoparticle of claim 1, wherein the SERS-active reporter molecule offormula A-Y is adsorbed on an outer surface of the nanoparticle.
 4. TheSERS active nanoparticle of claim 1, wherein the SERS-active reportermolecule of formula A-Y is covalently bound to an outer surface of thenanoparticle.
 5. The SERS active nanoparticle of claim 1, wherein thenanoparticle comprises a metal selected from the group consisting of Au,Ag, Cu, Na, Al, and Cr.
 6. The SERS active nanoparticle of claim 1,wherein the nanoparticle comprises an alloy of at least two metalsselected from the group consisting of Au, Ag, Cu, Na, Al, and Cr.
 7. TheSERS active nanoparticle of claim 1, wherein the nanoparticle has adiameter less than about 200 nm.
 8. The SERS active nanoparticle ofclaim 1, wherein the nanoparticle has a diameter between about 40 nm toabout 100 nm.
 9. The SERS active nanoparticle of claim 1, wherein theSERS-active reporter molecule of formula A-Y forms a layer on an outersurface of the nanoparticle, wherein the layer at least partially coversthe outer surface of the nanoparticle and is defined by an inner surfaceand an outer surface.
 10. The SERS active nanoparticle of claim 9,wherein the layer of the SERS-active reporter molecule of formula A-Yformed on the outer surface of the nanoparticle is selected from thegroup consisting of a submonolayer, a monolayer, and a multilayer. 11.The SERS active nanoparticle of claim 10, further comprising anencapsulant, wherein the encapsulant is disposed on at least one of theouter surface of the nanoparticle and the outer surface of the layer ofthe SERS-active reporter molecule of formula A-Y.
 12. The SERS activenanoparticle of claim 11, wherein the encapsulant comprises one or morematerials selected from the group consisting of a glass, a polymer, ametal, a metal oxide, and a metal sulfide.
 13. The SERS activenanoparticle of 12, wherein the glass comprises SiO_(x).
 14. The SERSactive nanoparticle of claim 12 wherein the encapsulant has a thicknessof about 1 nm to about 40 nm. 15-44. (canceled)
 45. A kit comprising areagent comprising one or more surface-enhanced Raman spectroscopy(SERS)-active nanoparticles having associated therewith at least oneSERS-active reporter molecule of Formula:A-Y wherein: A is:

wherein X₁ is CR₄ or N; Y is selected from the group consisting of:

wherein: r, s, and t are each independently an integer from 1 to 8; eachX₂ and X₃ is independently selected from the group consisting of C, S,and N, under the proviso that (i) when X₂ is C or S, R₅ is Z, or when X₃is C or S, R₆ is Z, as Z is defined herein below; (ii) if both X₂ and X₃are N at the same time, at least one of R₅ and R₆ is absent; and (iii)when X₂ is N, R₅ when present is Z′, or when X₃ is N, R₆ when present isZ′, wherein Z′ is selected from the group consisting of:—(CH₂)_(n)—X₄; —NR₈—(CH₂)_(p)—X₅; —(CH₂)_(q)X₆C(═O)—R₉,

wherein: n, p, q, u, and v are each independently an integer from 1 to8; X₄ and X₅ are each independently selected from the group consistingof hydroxyl, amino, and thiol; X₆ is O or NR₁₁; wherein: each R₁, R₂,R₃, R₄, R₅, R₆, R₈, R₁₀, R₁₁, and Z is independently selected from thegroup consisting of H, alkyl, substituted alkyl, heteroalkyl,substituted heteroalkyl, cycloalkyl, substituted cycloalkyl,cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl,aralkyl, hydroxyl, alkoxyl, hydroxyalkyl, hydroxycycloalkyl,alkoxycycloalkyl, aminoalkyl, acyloxyl, alkylaminoalkyl, andalkoxycarbonyl; R₇ is Z′; R₉ is —(CH₂)_(m)—X₇ or —(CH₂)_(m)—B, wherein mis an integer from 1 to 8; X₇ is halogen; and B is a binding memberhaving a binding affinity for a ligand or analyte to be detected. 46.The kit of claim 45, further comprising one or more of a samplecollection device, magnetic capture particles, a buffer solution, andcombinations thereof.
 47. The kit of claim 46, wherein the reagent isdisposed in the sample collection device.
 48. The kit of claim 45,wherein the one or more surface-enhanced Raman spectroscopy(SERS)-active nanoparticles comprise a plurality of SERS-activenanoparticles, each having associated therewith a SERS-active reportermolecule having a distinguishable SERS response, and wherein at leastone of the SERS-active reporter molecules comprises a SERS-activereporter molecule of Formula A-Y.